U.S. patent number 10,167,339 [Application Number 15/501,526] was granted by the patent office on 2019-01-01 for antagonistic anti-ox40l antibodies and methods of their use.
This patent grant is currently assigned to Baylor Research Institute. The grantee listed for this patent is BAYLOR RESEARCH INSTITUTE. Invention is credited to Patrick Blanco, Haruyuki Fujita, Shino Hanabuchi, Hyemee Joo, Yong-Jun Liu, SangKon Oh, Hideki Ueno, Sandra Zurawski.
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United States Patent |
10,167,339 |
Liu , et al. |
January 1, 2019 |
Antagonistic anti-OX40L antibodies and methods of their use
Abstract
Described herein are methods and compositions for treating
autoimmunity and inflammatory conditions without non-specific
suppression of the host immune system. In particular, the
anti-OX40L antibodies described herein are unique in that they not
only inhibit the differentiation of inflammatory T cells but also
promote the generation and function of regulatory T cells by
inducing IL-10 and inhibiting TNF-.alpha. and by reducing aberrant
Th2 cell responses. Furthermore, the methods and compositions
described herein eliminate or reduce aberrant T follicular helper
cell--(Tfh) responses that may contribute to the pathogenicity of
autoimmune disease.
Inventors: |
Liu; Yong-Jun (Gaithersburg,
MD), Zurawski; Sandra (Midlothian, TX), Oh; SangKon
(Baltimore, MD), Hanabuchi; Shino (Gaithersburg, MD),
Fujita; Haruyuki (Gaithersburg, MD), Ueno; Hideki
(Plano, TX), Blanco; Patrick (Verdelais, FR), Joo;
Hyemee (Dallas, TX) |
Applicant: |
Name |
City |
State |
Country |
Type |
BAYLOR RESEARCH INSTITUTE |
Dallas |
TX |
US |
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Assignee: |
Baylor Research Institute
(Dallas, TX)
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Family
ID: |
55264394 |
Appl.
No.: |
15/501,526 |
Filed: |
August 3, 2015 |
PCT
Filed: |
August 03, 2015 |
PCT No.: |
PCT/US2015/043408 |
371(c)(1),(2),(4) Date: |
February 03, 2017 |
PCT
Pub. No.: |
WO2016/022468 |
PCT
Pub. Date: |
February 11, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170349661 A1 |
Dec 7, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62032959 |
Aug 4, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
1/04 (20180101); A61P 21/00 (20180101); A61P
37/02 (20180101); A61P 29/00 (20180101); A61P
43/00 (20180101); A61P 19/02 (20180101); C07K
16/2875 (20130101); A61P 25/00 (20180101); A61P
11/06 (20180101); A61P 17/00 (20180101); A61P
37/06 (20180101); A61P 37/08 (20180101); A61P
9/10 (20180101); A61P 3/10 (20180101); C07K
2317/24 (20130101); A61K 2039/505 (20130101); C07K
2317/565 (20130101); A61K 2039/545 (20130101); C07K
2317/76 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/28 (20060101); A61K
39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0978287 |
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Feb 2000 |
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EP |
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WO 2007/133290 |
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Nov 2007 |
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WO |
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WO 2011/073180 |
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Jun 2011 |
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WO |
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Other References
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Primary Examiner: Shafer; Shulamith H
Attorney, Agent or Firm: Norton Rose Fulbright US LLP
Government Interests
The invention was made with government support under Grant Nos. U19
AI057234, U19 AI082715, and U19 AI089987 awarded by the National
Institutes of Health (NIH). The government has certain rights in
the invention.
Parent Case Text
This application is a national phase under 35 U.S.C. .sctn. 371 of
International Application No. PCT/US2015/043408, filed Aug. 3,
2015, which claims the benefit of priority to U.S. Provisional
Patent Application Ser. No. 62/032,959, filed Aug. 4, 2014, the
entire contents of each of which are hereby incorporated by
reference in their entirety.
Claims
What is claimed is:
1. A method for treating an autoimmune disease, inflammation,
and/or inflammation associated with an autoimmune disease, wherein
the disease or inflammation is due, at least in part, to pathogenic
Tfh cell responses, in a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
an OX40L inhibitor wherein the OX40L inhibitor comprises an OX40L
antibody or OX40L antigen-binding fragment comprising one of: a) a
heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 5, SEQ
ID NO: 6, and SEQ ID NO: 7 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; b) a
heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 19, SEQ
ID NO: 20, and SEQ ID NO: 21 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or c)
a heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 33, SEQ
ID NO: 34, and SEQ ID NO: 35 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.
2. The method of claim 1, wherein the disease or inflammation is
allergic disease asthma, atopic dermatitis, experimental autoimmune
encephalomyelitis, inflammatory bowel disease, contact
hypersensitivity, asthmatic airway hyperreaction, autoimmune
diabetes, atherosclerosis, systemic lupus erythematosus, rheumatoid
arthritis, multiple sclerosis, ulcerative colitis, graft versus
host disease, graft rejection, or polymyositis.
3. The method of claim 1, wherein the subject is a human.
4. The method of claim 1, wherein the anti-OX40L antibody is
neutralizing.
5. The method of claim 1, wherein the antibody is a human antibody,
humanized antibody, recombinant antibody, chimeric antibody, an
antibody derivative, a veneered antibody, a diabody, a monoclonal
antibody, or a polyclonal antibody.
6. The method of claim 5, wherein the antibody is a humanized
antibody.
7. The method of claim 1, wherein the antibody or antigen binding
fragment thereof comprises a modification.
8. The method of claim 1, wherein the OX40L inhibitor comprises an
antibody selected from the group consisting of 5C6, 19A3, and 44F3,
or a humanized or chimeric form thereof.
9. A method for treating systemic lupus erythematosus comprising
administering to the subject a therapeutically effective amount of
an OX40L inhibitor wherein the OX40L inhibitor comprises an OX40L
antibody or OX40L antigen-binding fragment comprising one of: a) a
heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 5, SEQ
ID NO: 6, and SEQ ID NO: 7 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; b) a
heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 19, SEQ
ID NO: 20, and SEQ ID NO: 21 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or c)
a heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 33, SEQ
ID NO: 34, and SEQ ID NO: 35 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.
10. The method of claim 9, wherein the subject is a human.
11. The method of claim 9, wherein the OX40L inhibitor comprises an
antibody selected from the group consisting of 5C6, 19A3, and 44F3,
or a humanized or chimeric form thereof.
12. A method for preventing, treating, or reducing the symptoms of
graft versus host disease in a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
an OX40L inhibitor wherein the OX40L inhibitor comprises an OX40L
antibody or OX40L antigen-binding fragment comprising one of: a) a
heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 5, SEQ
ID NO: 6, and SEQ ID NO: 7 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; b) a
heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 19, SEQ
ID NO: 20, and SEQ ID NO: 21 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or c)
a heavy chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 33, SEQ
ID NO: 34, and SEQ ID NO: 35 and a light chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.
13. The method of claim 12, wherein the subject is one that will
receive or has received transplanted tissues.
14. The method of claim 12, wherein the OX40L inhibitor comprises
an antibody selected from the group consisting of 5C6, 19A3, and
44F3, or a humanized or chimeric form thereof.
15. The method of claim 12, wherein the subject is a human.
16. The method of claim 12, wherein the method is for treating or
reducing the symptoms of graft versus host disease.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of medicine.
More particularly, it concerns pharmaceutical compositions for
treating autoimmune and inflammatory disorders and modifying immune
responses.
2. Description of Related Art
Autoimmune diseases and some inflammatory disorders arise from an
abnormal immune response of the body against substances and tissues
normally present in the body (autoimmunity or auto-inflammatory).
This may be restricted to certain organs (e.g. in autoimmune
thyroiditis) or involve a particular tissue in different places
(e.g. Goodpasture's disease which may affect the basement membrane
in both the lung and the kidney). Autoimmune diseases affect up to
50 million people in America alone, and the cause of autoimmunity
remains unknown. Furthermore, there are many inflammatory diseases
that are not associated with autoimmunity and may be idiopathic or
associated with a chronic or acute disorder.
The treatment of autoimmune and inflammatory diseases is typically
with immunosuppression or anti-inflammatants--medication that
decreases the immune and/or inflammation response. Conventional
immunetherapies using immunosuppressants, such as cyclosporine,
tacroliums, methotrexate or anti-TNFa/IL-6 non-specifically
suppress the function of T cell including non-pathogenic T cells in
the host. Therefore, treatment with these immunesuppressants often
results in the development of severe infections and sometimes leads
to the lethal consequences. There is a need in the art for
therapeutics that treat autoimmune and/or inflammatory responses
without global immunosuppression.
SUMMARY OF THE INVENTION
Described herein are methods and compositions for treating
autoimmunity and inflammatory conditions without non-specific
suppression of the host immune system. In particular, the
anti-OX40L antibodies described herein are unique in that they not
only inhibit the differentiation of inflammatory T cells but also
promote the generation and function of regulatory T cells by
inducing IL-10 and inhibiting TNF-.alpha. and by reducing aberrant
Th2 cell responses. Furthermore, the methods and compositions
described herein eliminate or reduce aberrant T follicular helper
cell--(Tfh) responses that may contribute to the pathogenicity of
autoimmune disease.
Disclosed is a pharmaceutical composition comprising an isolated
anti-OX40L antibody or antigen-binding fragment thereof comprising
a heavy chain variable domain comprising three complementarity
determining region (CDR) amino acid sequences, wherein one or more
of the CDRs comprise an amino acid sequence selected from SEQ ID
NOs: 5-7, 19-21, 33-35, or an equivalent thereof. In some
embodiments, an antibody of antigen binding fragment comprises
three CDRs from a variable domain. In some embodiments, the three
heavy chain variable domain CDRs comprise each of the amino acid
sequences of SEQ ID NOs: 5-7 or an equivalent thereof. In other
embodiments, the three heavy chain variable domain CDRs each
comprise the amino acid sequence of SEQ ID NOs: 19-21 or an
equivalent thereof. In other embodiments, the three heavy chain
variable domain CDRs each comprise the amino acid sequence of SEQ
ID NOs: 33-35 or an equivalent thereof. In further embodiments, the
anti-OX40L antibody or antigen binding fragment thereof further
comprises a light chain variable domain comprising three
complementarity determining region (CDR) amino acid sequences,
wherein one or more of the CDRs comprise an amino acid sequence
selected from SEQ ID NOs: 12-14, 26-28, 40-42, or an equivalent
thereof. In some embodiments, there are three light chain variable
domain CDRs that each comprise the amino acid sequence of SEQ ID
NOs: 12-14 or an equivalent thereof. In other embodiments, there
are three light chain variable domain CDRs that each comprise the
amino acid sequence of SEQ ID NOs: 26-28 or an equivalent thereof.
In further embodiments, there are three light chain variable domain
CDRs that each comprise the amino acid sequence of SEQ ID NOs:
40-42 or an equivalent thereof.
In certain embodiments described herein, the anti-OX40L antibody or
antigen binding fragment thereof binds to human OX40L or an
equivalent thereof. In a specific embodiment, the OX40L antibody is
a neutralizing antibody that disrupts, prevents, or impedes a
function or interaction (e.g. OX40-OX40L interaction) of the OX40L
protein. In particular embodiments, the anti-OX40L antibody
comprises the 5C6, 19A3, or 44F3 monoclonal antibody.
In other embodiments, the antibody is a human antibody, humanized
antibody, recombinant antibody, chimeric antibody, an antibody
derivative, a veneered antibody, a diabody, an engineered antibody,
a multi-specific antibody, a DARPin (designed ankyrin repeat
protein), a monoclonal antibody, or a polyclonal antibody. In some
embodiments, the antibody is a humanized antibody.
In further embodiments, the OX40L antibody comprises a
modification. In certain embodiments, the modification is a
conservative amino acid mutation within the VH and/or VL CDR 1, CDR
2 and/or CDR 3 regions; of conservative amino acid mutations in the
Fc hinge region; pegylation. conjugation to a serum protein;
conjugation to human serum albumin; conjugation to a detectable
label; conjugation to a diagnostic agent; conjugation to an enzyme;
conjugation to a fluorescent, luminescent, or bioluminescent
material; conjugation to a radioactive material; or conjugation to
a therapeutic agent.
Certain embodiments relate to a pharmaceutical composition
comprising an isolated humanized IgG anti-OX40L antibody or
antigen-binding fragment thereof comprising a heavy chain variable
domain comprising the complementarity determining region (CDR)
amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7 and a light chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 12, SEQ
ID NO: 13, and SEQ ID NO: 14.
Other embodiments relate to a pharmaceutical composition comprising
an isolated humanized IgG anti-OX40L antibody or antigen-binding
fragment thereof comprising a heavy chain variable domain
comprising the complementarity determining region (CDR) amino acid
sequences of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 and a
light chain variable domain comprising the complementarity
determining region (CDR) amino acid sequences of SEQ ID NO: 26, SEQ
ID NO: 27, and SEQ ID NO: 28.
Further embodiments relate to a pharmaceutical composition
comprising an isolated humanized IgG anti-OX40L antibody or
antigen-binding fragment thereof comprising a heavy chain variable
domain comprising the complementarity determining region (CDR)
amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID
NO: 35 and a light chain variable domain comprising the
complementarity determining region (CDR) amino acid sequences of
SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.
Further aspects of the disclosure relate to an isolated
polynucleotide comprising a nucleic acid sequence encoding a
polypeptide chain of an anti-OX40L antibody or antigen-binding
fragment thereof comprising a heavy chain variable domain
comprising three complementarity determining region (CDR) amino
acid sequences, wherein one or more of the CDRs comprise an amino
acid sequence selected from SEQ ID NOs: 5-7, 19-21, 33-35, or an
equivalent thereof. A further aspect relates to an isolated
polynucleotide comprising a nucleic acid sequence encoding a
polypeptide chain of an anti-OX40L antibody or antigen-binding
fragment thereof comprising a light chain variable domain
comprising three complementarity determining region (CDR) amino
acid sequences, wherein one or more of the CDRs comprise an amino
acid sequence selected from SEQ ID NOs: 12-14, 26-28, 40-42, or an
equivalent thereof. Also disclosed is an expression vector
comprising a polynucleotide described herein and a host cell
comprising a polynucleotide described herein operably linked to a
regulatory sequence.
Embodiments are provided in which the anti-OX40L antibody or
antigen-binding fragment thereof comprises one or more CDR domains
from an antibody that specifically binds to OX40L. In particular
embodiments, the anti-OX40L antibody or antigen-binding fragment
thereof comprises one, two, three, four, five, six, or more CDR
domains from among the VH or VL domain of the 19A3, 5C6, and 44F3
monoclonal antibodies. In certain aspects, the anti-OX40L antibody
or antigen-binding fragment thereof comprises six CDR domains from
among the VH or VL domains of the 19A3, 5C6, and 44F3 monoclonal
antibodies. In some embodiments, the anti-OX40L antibody or
antigen-binding fragment thereof comprises a sequence at least or
at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% (or any range
derivable therein) identical to the VH or VL domain of the 19A3,
5C6, and 44F3 monoclonal antibodies. Embodiments are provided in
which the anti-OX40L antibody or antigen-binding fragment thereof
comprises the VH domain from the 19A3, 5C6, or 44F3 monoclonal
antibody and/or the VL domain the 19A3, 5C6, or 44F3 monoclonal
antibody.
In certain embodiments, the antibody or antigen-binding fragment
thereof is recombinant. In certain aspects, the recombinant
polypeptide comprises at least 90%, 95%, or 99% of one or more CDR
domains from the VH or VL domain of the 19A3, 5C6, and 44F3
monoclonal antibodies. In some embodiments, the recombinant
polypeptide comprises two, three, four, five, six, or more CDR
domains from the VH or VL domain of the 19A3, 5C6, and 44F3
monoclonal antibodies.
In some embodiments, a recombinant polypeptide comprises i) CDR1,
CDR2, and/or CDR3 from the variable light chain of 19A3 (SEQ ID
NOS:12-14); and/or ii) CDR1, CDR2, and/or CDR3 from the variable
heavy chain of 19A3 (SEQ ID NOS:5-7). In some embodiments, a
recombinant polypeptide comprises i) CDR1, CDR2, and/or CDR3 from
the variable light chain of 5C6 (SEQ ID NOS:26-28); and/or ii)
CDR1, CDR2, and/or CDR3 from the variable heavy chain of 5C6 (SEQ
ID NOS:19-21). In some embodiments, a recombinant polypeptide
comprises i) CDR1, CDR2, and/or CDR3 from the variable light chain
of 44F3 (SEQ ID NOS:40-42); and/or ii) CDR1, CDR2, and/or CDR3 from
the variable heavy chain of 44F3 (SEQ ID NOS:33-35). The sequences
for these CDRs can be found in the disclosure that follows.
In some embodiments, there is a purified polypeptide comprising one
or more anti-OX40L antibody CDR domains. As indicated above, the
polypeptide may comprise 1, 2, 3, 4, 5, or 6 CDRs from the light
and/or heavy chain variable regions of an anti-OX40L antibody. In
certain embodiments, a polypeptide contains CDR1, CDR2, and/or CDR3
from the light chain variable region of a particular antibody. It
is contemplated that while in some embodiments a polypeptide has a
CDR1, CDR2, and CDR3 from the variable region of a light chain
and/or the variable region of a heavy chain that the CDR1, CDR2,
and CDR3 need not be from the same antibody. While some
polypeptides have CDR1, CDR2, and CDR3 from the same antibody or
based on the same antibody, it is contemplated that a CDR1 from one
antibody may be substituted with a CDR from or based on another
antibody. For example, a polypeptide may comprise a CDR1 from or
based on the light chain variable region of 19A3, a CDR2 from or
based on the light chain variable region of 19A3, but have a CDR3
from or based on the variable light chain region of 5C6. It is
generally contemplated, however, that when a single set of CDR1,
CDR2, and CDR3 are employed together that they all be from a light
chain variable region or from a heavy chain variable region, but
not a mix from both.
Alternatively, the polypeptide may contain a CDR1 sequence that is,
is at most or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99,
100% identical (or any range derivable therein) to the entire
sequence set forth in SEQ ID NOs:12, 26, and 40, which are CDR1
sequences from the light chain variable region of an anti-OX40L
antibody. Alternatively or additionally, the polypeptide may
contain a CDR2 sequence that is, is at most or is at least 70, 75,
80, 85, 90, 95, 96, 97, 98, 99, 100% identical (or any range
derivable therein) to the entire sequence set forth in SEQ ID
NOs:13, 27, and 41, which are CDR2 sequences from the light chain
variable region of an anti-OX40L antibody. Alternatively or
additionally, the polypeptide may contain a CDR3 sequence that is,
is at most or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99,
100% identical (or any range derivable therein) to the entire
sequence set forth in SEQ ID NOs:14, 28, and 42, which are CDR3
sequence from the light chain variable region of an anti-OX40L
antibody. Alternatively or additionally, the polypeptide may
contain a CDR1 sequence that is, is at most or is at least 70, 75,
80, 85, 90, 95, 96, 97, 98, 99, 100% identical (or any range
derivable therein) to the entire sequence set forth in SEQ ID NOs:
5, 19, and 33, which are CDR1 sequences from the heavy chain
variable region of an anti-OX40L antibody. Alternatively or
additionally, the polypeptide may contain a CDR2 sequence that is,
is at most or is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99,
100% identical (or any range derivable therein) to the entire
sequence set forth in SEQ ID NOs:6, 20, and 34, which are CDR2
sequences from the heavy chain variable region of an anti-OX40L
antibody. Alternatively or additionally, the polypeptide may
contain a CDR3 sequence that is, is at most or is at least 70, 75,
80, 85, 90, 95, 96, 97, 98, 99, 100% identical (or any range
derivable therein) to the entire sequence set forth in SEQ ID
NOs:7, 21, and 35, which are CDR3 sequences from the heavy chain
variable region of an anti-OX40L antibody.
Method aspects of the disclosure relate to a method for treating or
preventing inflammation in a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
an OX40L inhibitor.
Further aspects relate to a method for treating or preventing an
autoimmune disease in a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
an OX40L inhibitor.
In certain aspects, the method is for preventing inflammation
associated with an autoimmune disease in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of an OX40L inhibitor.
Other aspects relate to a method for reducing inflammatory Th2 cell
responses, for increasing IL-10 production and/or for reducing
TNF-a production in a subject in need thereof comprising
administering to the subject a therapeutically effective amount of
an OX40L inhibitor. In some embodiments, the inflammatory Th2 cell
responses comprise IL-10 low/TNF-.alpha. height producing
inflammatory Th2 cells.
Other aspects relate to a method for decreasing pathogenic Tfh cell
responses in a subject in need thereof comprising administering a
therapeutically effective amount of an OX40L inhibitor.
In some embodiments, the subject being treated is one that has an
autoimmune disease. In certain aspects, the autoimmune disorder in
the subject is treated by the administration of an OX40L inhibitor,
which decreases pathogenic Tfh cell responses in the subject.
In further embodiments, the subject has inflammation. The
inflammation in the subject may be reduced or eliminated by
administering an OX40L inhibitor, which increases IL-10 production
and reduces TNF-a production in the subject. The OX40L inhibitor
may also reduce inflammation by reducing inflammatory Th2 cell
responses in the subject.
In some embodiments, the subject being treated has an autoimmune
disorder or has inflammation as a result of an autoimmune disorder.
In some embodiments, the autoimmune disease selected from the group
allergic disease asthma, atopic dermatitis, experimental autoimmune
encephalomyelitis, inflammatory bowel disease, contact
hypersensitivity, asthmatic airway hyperreaction, autoimmune
diabetes, atherosclerosis, systemic lupus erythematosus, Sjogren's
syndrome, type 1 diabetes, rheumatoid arthritis, multiple
sclerosis, ulcerative colitis, polymyositis, mixed connective
tissue disease, systemic sclerosis, myasthenia gravis, thyroiditis,
autoimmune hemolytic anemia, immune thrombocytopenic purpura,
dermatomyositis, antineutrophil cytoplasmic autoantibody-mediated
disease, IgA-mediated vasculitis, and Ig4-related disorders. In
some embodiments, the autoimmune disease is systemic lupus
erythematosus.
In further embodiments, the inflammation may be idiopathic. In yet
further embodiments, the inflammation may be the result of a
disease or condition that is not autoimmune related, such as an
injury.
Further aspects relate to a method for treating or preventing graft
versus host disease or graft rejection in a subject in need thereof
comprising administering to the subject a therapeutically effective
amount of an OX40L inhibitor.
Graft-versus-host disease (GVHD) is a common complication following
an allogeneic tissue transplant. It is commonly associated with
stem cell or bone marrow transplant but the term also applies to
other forms of tissue graft. Immune cells (white blood cells) in
the tissue (the graft) recognize the recipient (the host) as
"foreign". The transplanted immune cells then attack the host's
body cells. GVHD may also occur after a blood transfusion if the
blood products used have not been irradiated.
Graft rejection occurs when transplanted tissue is rejected by the
recipient's immune system, which destroys the transplanted tissue.
Graft rejection may also be referred to as transplant rejection or
host versus graft disease.
In some embodiments of any of the above-disclosed methods, the
subject is one that will receive or has received transplanted
tissues. In a related embodiment, the transplanted tissue is an
allograft. An allograft (also known as allotransplantation,
allogeneic transplant, or homograft) is the transplantation of
cells, tissues, or organs, to a recipient from a genetically
non-identical donor of the same species. In a related embodiment,
the subject is one that has a complication from the transplanted
tissue, wherein the complication is graft rejection or GVHD.
The term "subject," "individual" or "patient" is used
interchangeably herein and refers to a vertebrate, for example a
primate, a mammal or preferably a human. Mammals include, but are
not limited to equines, canines, bovines, ovines, murines, rats,
simians, humans, farm animals, sport animals and pets. In one
embodiment of the methods described herein, the subject is a human
subject.
The OX40L inhibitor may be an siRNA, dsRNA, miRNA, ribozyme,
molecular inhibitor, small molecule, antibody, or antigen binding
fragment. In some embodiments, the OX40L inhibitor is an OX40L
antibody or antigen-binding fragment thereof. In further
embodiments, the OX40L inhibitor comprises a composition as
described herein. In yet further embodiments, the OX40L inhibitor
comprises a polypeptide, polynucleotide, antibody, host cell, or
expression vector described herein.
As used herein the specification, "a" or "an" may mean one or more.
As used herein in the claim(s), when used in conjunction with the
word "comprising", the words "a" or "an" may mean one or more than
one.
The use of the term "or" in the claims is used to mean "and/or"
unless explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
Throughout this application, the term "about" is used to indicate
that a value includes the inherent variation of error for the
device, the method being employed to determine the value, or the
variation that exists among the study subjects.
Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
FIG. 1 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 5C6 (AB104_105.5C6.3F9)
anti-OX40L mAb, ik-5 anti-OX40L mAb, or control antibody (IgG2a)
according to methods described in Example 1. The production of
IL-4, IL-5, IL-10, IL-13, TNF-.alpha. and IFN-.gamma. were measured
by ELISA.
FIG. 2 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 19A3
(AB104_105.19A3.2C4) anti-OX40L mAb, ik-5 anti-OX40L mAb, or
control antibodies (IgG2a and IgG2b) according to methods described
in Example 1. The production of IL-4, IL-5, IL-10, IL-13,
TNF-.alpha. and IFN-.gamma. were measured by ELISA.
FIG. 3 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 5C6 (AB104_105.5C6.3F9)
anti-OX40L mAb, 44F3 (AB104_105.44F3.2F7) anti-OX40L mAb, 19A3
(AB104_105.19A3.2C4) anti-OX40L mAb, ik-5 anti-OX40L mAb, or
control antibodies (IgG2a and IgG2b) according to methods described
in Example 1. The production of IL-4, IL-5, IL-10, IL-13,
TNF-.alpha. and IFN-.gamma. were measured by ELISA.
FIG. 4 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 5C6 (AB104_105.5C6.3F9)
anti-OX40L mAb, ik-5 anti-OX40L mAb, or control antibody (IgG2a)
according to methods described in Example 1. Cells were harvested,
and the proliferation and viability of the cells was
determined.
FIG. 5 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 19A3
(AB104_105.19A3.2C4) anti-OX40L mAb, ik-5 anti-OX40L mAb, or
control antibodies (IgG2a and IgG2b) according to methods described
in Example 1. Cells were harvested, and the proliferation and
viability of the cells was determined.
FIG. 6 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 5C6 (AB104_105.5C6.3F9)
anti-OX40L mAb, 44F3 (AB104_105.44F3.2F7) anti-OX40L mAb, 19A3
(AB104_105.19A3.2C4) anti-OX40L mAb, ik-5 anti-OX40L mAb, or
control antibodies (IgG2a and IgG2b) according to methods described
in Example 1. Cells were harvested, and the proliferation and
viability of the cells was determined.
FIG. 7 depicts the results of an assay in which Dendritic cells and
T cells were cocultured in the presence of 19A3
(AB104_105.19A3.2C4) or control antibody (Mouse IgG2b) according to
methods described in Example 1. The production of IL-10 and
TNF-.alpha. were measured by ELISA.
FIG. 8 shows a repeat of the experiment described in FIG. 7.
FIG. 9A-9C shows that increased OX40L expression by myeloid APCs in
inflammatory tonsils. (A) Expression of OX40L, ICOSL, 4-1BBL and
GITRL on myeloid CD11c.sup.+HLA-DR.sup.+ APCs from pediatric
tonsils. A representative result out of 9 independent experiments.
(B) Frequency of OX40L.sup.+, ICOSL.sup.+, GITRL.sup.+, and
4-1BBL.sup.+ cells within tonsillar myeloid APCs. Mean.+-.s.d.,
n=9. One way ANOVA. *** p<0.001. (C) OX40L.sup.+CD11c.sup.+ APCs
in inflammatory tonsils. GC: germinal center; MZ: mantle zone; Epi:
Epithelial layers. The scale bars on the top and the bottom panels
shows 100 .mu.m and 10 .mu.m, respectively.
FIG. 10A-10E shows that OX40L expression by myeloid APCs from SLE
patients. (A) OX40L.sup.+ myeloid APCs in skin and kidney biopsies
from adult SLE patients and subjects without autoimmune diseases. A
representative result of 5 skin and 3 kidney biopsy samples from
SLE patients and 5 skin and 2 kidney biopsy samples from controls.
Scale bar=100 .mu.m. (B) Representative flow data on OX40L
expression by blood myeloid CD11c.sup.+HLA-DR.sup.+ APCs from the
three groups: healthy donors (HD), inactive (iSLE) and active
(aSLE) SLE patients. (C) Frequency of OX40L.sup.+ cells within
blood myeloid APCs in the three groups in adult and pediatric
cohorts. Top: the adult cohort; 16 HD, 38 iSLE, and 31 aSLE
samples. Bottom: the children cohort; 14 HD, 20 iSLE, and 14 aSLE
samples. One-way ANOVA. * p<0.05, ** p<0.01, *** p<0.001.
(D) Correlation between the percentage of OX40L.sup.+ cells within
CD11c+HLA-DR.sup.+ myeloid APCs (adults: n=69 and children: n=38)
and disease activity assessed by the SLEDAI. Statistical analysis
was performed with the Spearman test. (E) Composition of blood
OX40L.sup.+ myeloid APCs by different subsets
(CD14.sup.+CD16.sup.-, CD14.sup.+CD16.sup.+, CD14.sup.-CD16.sup.-,
CD14.sup.-CD16.sup.+) in adult (n=28) and pediatric (n=34) SLE
patients. Mean.+-.s.d.
FIG. 11A-11C shows that OX40 signals induce upregulation of Tfh
genes. (A) Tfh gene expression by naive and memory Th cells (from
three donors) activated with anti-CD3 and anti-CD28 in the presence
or absence of sOX40L for 48 h. Transcript counts in the cultured Th
cells are shown after normalization. Mean.+-.s.d, n=3. Paired
t-test. * p<0.05, ** p<0.01. (B) Tfh gene expression profiles
by naive and memory Th cells activated with anti-CD3 and anti-CD28
in the presence of indicated reagents for 48 h. Transcript counts
in Th cells cultured in the presence of the indicated reagents were
normalized to those in control Th cells in each donor. (C)
Transcript counts in memory Th cells activated with anti-CD3 and
anti-CD28 in the presence of indicated reagents. The bars in each
bar graph represent, from left to right, "none," "OX40L," "IL-2,"
"IL-2+OX40L," and "IFN-.gamma.." Mean.+-.s.d., n=3. One-way ANOVA.
* P<0.05, ** P<0.01, *** P<0.001.
FIG. 12A-12C shows that OX40L stimulation promotes the
differentiation of naive and memory T cells into Tfh-like cells.
(A) Expression of CXCR5, IL-21 and CD40L by naive and memory Th
cells activated for 4 d with anti-CD3 and anti-CD28 in the presence
or absence of sOX40L and/or IL-12. Gated to FSCh.sup.hiSSC.sup.hi
activated cells. A representative result out of 3 independent
experiments is shown. (B) Frequency of CXCR5.sup.+IL-21.sup.+ and
CXCR5.sup.+CD40L.sup.+ cells developed in naive or memory Th cells
after activation with anti-CD3 and anti-CD28 in the presence or
absence of sOX40L and/or IL-12. One-way ANOVA. * p<0.05, **
p<0.01, *** p<0.001, n=3. (C) Naive or memory Th cells were
activated for 4 d with anti-CD3 and anti-CD28 in the presence of
sOX40L and/or IL-12, and then cultured with autologous memory B
cells. IgG concentrations in the supernatant of each well are
shown. A representative result out of 2 independent experiments is
shown.
FIG. 13A-13F shows that blood CD14.sup.+ APCs in human SLE promote
the generation of Tfh-like cells via OX40L. (A) Frequency of
CXCR5.sup.+IL-21.sup.+ cells (among FSC.sup.hiSSC.sup.hi activated
cells) developed in naive Th cells after culture for 7 d with
allogeneic CD14.sup.+ APCs from inactive (iSLE, n=5) and active
(aSLE, n=5) SLE patients. Mann-Whitney U-test. ** p<0.01. (B)
Expression of CXCR5 and IL-21 by naive Th cells cultured with
allogeneic SLE CD14.sup.+ APCs in the presence of an OX40L
neutralizing mAb or a control IgG. A representative result out of 5
experiments is shown. (C) Decreased generation of
CXCR5.sup.+IL-21.sup.+ cells (among FSC.sup.hiSSC.sup.hi activated
cells) by anti-OX40L. Results with APCs from active SLE patients
are shown. Paired t-test. ** p<0.01. (D) Correlation between the
frequency of OX40L.sup.+ cells within the CD14.sup.+ APCs and the
frequency of CXCR5.sup.+IL-21.sup.+ Th cells generated in the
cultures. Spearman correlation test, n=10. (E) Expression of ICOS
on blood Tfh cells in the three groups; aSLE, iSLE, and HD. A
representative flow result is shown. (F) Correlation between the
frequency of OX40L.sup.+ cells within blood myeloid APCs and the
frequency of ICOS.sup.+ cells within blood Tfh cells in SLE
patients. Spearman correlation test, n=19.
FIG. 14A-14E shows that RNP/anti-RNP ICs promote OX40L expression
by myeloid APCs in a TLR7-dependent manner. (A) Expression of OX40L
(MFI) by purified normal monocytes exposed to control sera (n=7) or
SLE sera (n=21). Mann-Whitney U-test. ** p<0.01. A
representative staining is shown on the left panel. (B) OX40L
expression upon stimulation of purified normal monocytes by TLR3,
TLR7 or TLR9 agonists. A representative staining out of 4 different
experiments is shown. (C) OX40L expression (MFI) in normal
monocytes exposed to SLE sera (n=7) in the presence or not of a
TLR7 inhibitor. Paired t-test. ** p<0.01. A representative
staining is shown on the left panel. (D) OX40L expression (MFI) in
normal monocytes exposed to anti-RNP.sup.neg SLE sera (n=5) or
anti-RNP.sup.pos SLE sera (n=16). Mann-Whitney U-test. **
p<0.01. (E) OX40L expression of purified normal monocytes
exposed to anti-RNP.sup.neg SLE serum (upper panel), the serum
supplemented with anti-RNP-containing IgG (medium panel), the serum
spiked with anti-RNP-containing IgG in the presence of a TLR7
inhibitor (lower panel). A representative staining out of three
independent experiments is shown.
FIG. 15 shows that OX40L expression by myeloid APCs in SLE.
Analysis of OX40L expression by blood CD11c.sup.+HLA-DR.sup.+ cells
in 15 healthy donor (HD), 37 SLE, 13 systemic sclerosis (SSc) and
11 rheumatoid arthritis (RA) patients. One-way ANOVA. ***
P<0.001.
FIG. 16A-16B demonstrates that blood myeloid APCs in active SLE
patients do not express ICOSL, 4-1BBL, or GITRL. Analysis of OX40L,
GITRL, ICOSL, 4-1BBL expression by blood myeloid APCs in 8 active
SLE patients. A representative flow result is shown in panel a. b.
One-way ANOVA. ** P<0.01, * P<0.05.
FIG. 17 demonstrates that a majority of blood OX40L.sup.+ myeloid
APCs express CD14. CD14 and CD16 expression was analyzed on blood
OX40L+ myeloid APCs in adult and pediatric SLE patients. A
representative flow result from 11 adult SLE and 12 pediatric SLE
patient samples is shown.
FIG. 18 shows that OX40L expression on blood myeloid APCs decreases
after treatment in adult SLE patients. The expression of OX40L on
blood myeloid APCs was analyzed in 11 flaring previously untreated
adult SLE patients before and after treatment (Tx). The percentage
of OX40L.sup.+ cells within blood myeloid APCs before and after
treatment is shown. Paired t-test, n=11.
FIG. 19 demonstrates that OX40 signals induce naive Th cells to
express Tfh genes. Tfh gene expression profiles by naive Th cells
activated with anti-CD3 and anti-CD28 in the presence of indicated
reagents for 48 h. The bars in each bar graph represent, from left
to right, "none," "OX40L," "IL-2," "IL-2+OX40L," and "IFN-.gamma.."
Mean.+-.s.d., n=3. One-way ANOVA. * P<0.05, ** P<0.01, ***
P<0.001.
FIG. 20 shows that OX40L stimulation promotes naive and memory Th
cells to acquire the phenotype of Tfh cells. Naive and memory Th
cells were cultured with anti-CD3 and CD28 in the presence or
absence of IL-12 and/or soluble OX40L. The phenotype of activated
(FSChiSSChi) cells was analysed by flow cytometry at day 5. A
representative flow result is shown in the top panel. The
percentage of CXCR5.sup.+ICOS.sup.+ and CXCR5.sup.+CCR7.sup.- cells
within activated cells in the different conditions is shown in the
bottom panel. One-way ANOVA. *** P<0.001, ** P<0.01, *
P<0.05.
FIG. 21 shows that OX40L stimulation induces naive and memory Th
cells to express IL-21, IL-2 and TNF-.alpha.. CXCR5, IL-21 and
CD40L expression of naive and memory Th cells activated with
anti-CD3 and anti-CD28 in the presence or absence of sOX40L.
Cultured Th cells were re-stimulated for 6 h with PMA and ionomycin
in the presence of brefeldin A and monensin to analyze the
intracytplasmic cytokines. Paired t-test. *** P<0.001, **
P<0.01, * P<0.05.
FIG. 22 shows that the percentage of OX40L.sup.+ cells within blood
myeloid APCs correlate with the frequency of blood Tfh cells in SLE
patients. The correlation between the frequency of OX40L.sup.+
cells within blood myeloid APCs and the frequency of blood Tfh
cells was analyzed in 19 SLE patients. Statistical analysis was
performed with the Spearman test.
FIG. 23 shows that the percentage of OX40L.sup.+ cells within blood
myeloid APCs does not correlate with the frequency of blood Th1,
Th2, and Th17 cells in SLE patients. Gating strategy for the
analysis of blood Th1, Th2, and Th17 cells (within memory
CXCR5.sup.- Th cells) is shown in the top panel. The correlation
between the frequency of OX40L.sup.+ cells within blood myeloid
APCs and the frequency of blood Th1, Th2, and Th17 cells was
analyzed in 19 SLE patients. Statistical analysis was performed
with the Spearman test.
FIG. 24 shows that OX40L expression by SLE sera is dependent on
RNA. OX40L expression by purified normal monocytes exposed to SLE
sera in the presence or not of RNAse (0.1 mg/ml. Qiagen). Results
with serum samples from 5 SLE patients. Paired t-test, ***
P<0.001.
FIG. 25 shows the percent survival of animals after transplantation
of graft tissue.
FIG. 26A-B shows that anti-OX40L antibody does not interfere with
human chimerism. Shown are FACS plots with IgG2b treated (FIG. 26A)
and anti-OX40L (FIG. 26B) treated mice.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Conventional immunetherapies using immunosuppressants, such as
cyclosporine, tacroliums, methotrexate or anti-TNFa/IL-6
non-specifically suppress T cell functions, including
non-pathogenic T cells in the host. Therefore, treatment with these
immunosuppressants often results in the development of severe
infections and sometimes leads to the lethal consequences. Methods
and compositions described herein are directed to the deliberate
blockade of the OX40-OX40L interaction and the specific suppression
of the recent activation of inflammatory T cells. In turn, the
differentiation of inflammatory T cells is converted into
regulatory T cells by the induction of IL-10 and inhibition of
TNF-a production without global immune suppression. Importantly,
the targeting of OX40L can modulate only antigen-specific T cell
repertoire without disrupting the function of the other T cell
repertoires, resulting in less immunosuppressive side effects.
Using unique screening methods, Applicants have made anti-human
OX40L neutralizing MAbs, which 1) recognize unique epitopes on
human OX40L; 2) inhibit the differentiation of IL-10 low/TNFa high
producing inflammatory Th2 primed by TSLP-mDCs; and 3) inhibit the
proliferation and the production of TNF-a, and promote IL-10 by CD4
T cells cultured with OX40L-transfected cell line. These OX40L
blocking monoclonal antibodies are powerful immune modulators and
provide promising therapeutics for the inflammatory diseases such
as graft versus host disease, system lupus erythematosus,
cardiovascular disease (e.g. atherosclerosis), and inflammatory
diseases such as those described herein.
I. OX40L Inhibitors
A. Antibodies
Methods and compositions of the disclosure relate to OX40L
inhibitors. The term "OX40L" refers to a protein that has been
found to be involved in T cell antigen-presenting cell (APC)
interactions. OX40L may also be known as TNFSF4, GP34, CD252,
OX40L, TXGP1, and CD134L. This protein is a ligand for OX40 (also
known as CD134, TNFRSF4, ACT35, IMD16, and TXGP1L). The human
protein sequence of OX40L is represented by Genbank Accession Nos:
NP_003317.1, P23510.1, BAB18304.1, and P43489.1. The sequences
associated with these accession numbers are specifically
incorporated by reference.
In certain embodiments, the OX40L inhibitor is an antibody or
antigen-binding fragment thereof. As used herein, an "antibody"
includes whole antibodies and any antigen binding fragment or a
single chain thereof. Thus the term "antibody" includes any protein
or peptide containing molecule that comprises at least a portion of
an immunoglobulin molecule. Examples of such include, but are not
limited to a complementarity determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework (FR) region or any portion thereof or at least
one portion of a binding protein. In certain embodiments, the
antibody or antigen binding fragment specifically binds human
OX40L.
The antibody can be any of the various antibodies described herein,
non-limiting, examples of such include a polyclonal antibody, a
monoclonal antibody, a chimeric antibody, a human antibody, a
veneered antibody, a diabody, a humanized antibody, an antibody
derivative, a recombinant antibody, a recombinant humanized
antibody, an engineered antibody, a multi-specific antibody, a
DARPin, or a derivative or fragment of each thereof.
Antibodies can be generated using conventional techniques known in
the art and are well-described in the literature. Several
methodologies exist for production of polyclonal antibodies. For
example, polyclonal antibodies are typically produced by
immunization of a suitable mammal such as, but not limited to,
chickens, goats, guinea pigs, hamsters, horses, mice, rats, and
rabbits. An antigen is injected into the mammal, induces the
B-lymphocytes to produce immunoglobulins specific for the antigen.
Immunoglobulins may be purified from the mammal's serum. Common
variations of this methodology include modification of adjuvants,
routes and site of administration, injection volumes per site and
the number of sites per animal for optimal production and humane
treatment of the animal. For example, adjuvants typically are used
to improve or enhance an immune response to antigens. Most
adjuvants provide for an injection site antigen depot, which allows
for a stow release of antigen into draining lymph nodes. Other
adjuvants include surfactants which promote concentration of
protein antigen molecules over a large surface area and
immunostimulatory molecules. Non-limiting examples of adjuvants for
polyclonal antibody generation include Freund's adjuvants, Ribi
adjuvant system, and Titermax. Polyclonal antibodies can be
generated using methods known in the art some of which are
described in U.S. Pat. Nos. 7,279,559; 7,119,179; 7,060,800;
6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.
Unless specified otherwise, the antibodies can be polyclonal or
monoclonal and can be isolated from any suitable biological source,
e.g., murine, rat, rabbit, goat, camelid, sheep or canine.
As used herein, "monoclonal antibody" refers to an antibody
obtained from a substantially homogeneous antibody population.
Monoclonal antibodies are highly specific, as each monoclonal
antibody is directed against a single determinant on the antigen.
The antibodies may be detectably labeled, e.g., with a
radioisotope, an enzyme which generates a detectable product, a
fluorescent protein, and the like. The antibodies may be further
conjugated to other moieties, such as members of specific binding
pairs, e.g., biotin (member of biotin-avidin specific binding
pair), and the like. The antibodies may also be bound to a solid
support, including, but not limited to, polystyrene plates or
beads, and the like. In some instances, the antibodies are
indirectly labeled. Indirect labelling may involve the labeling of
a protein that binds to the antibody, such as a secondary
antibody.
Monoclonal antibodies can be generated using conventional hybridoma
techniques known in the art and well-described in the literature.
For example, a hybridoma is produced by fusing a suitable immortal
cell line (e.g., a myeloma cell line such as, but not limited to,
Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, P3X63Ag8,653, Sp2 SA3,
Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MIA 144, ACT IV, MOLT4, DA-1,
JURKAT, WEHI, K-562, COS, RAJI, NIH 313, HL-60, MLA 144, NAMAIWA,
NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas,
fusion products thereof, or any cell or fusion cell derived there
from, or any other suitable cell line as known in the art, with
antibody producing cells, such as, but not limited to, isolated or
cloned spleen, peripheral blood, lymph, tonsil, or other immune or
B cell containing cells, or any other cells expressing heavy or
light chain constant or variable or framework or CDR sequences,
either as endogenous or heterologous nucleic acid, as recombinant
or endogenous, viral, bacterial, algal, prokaryotic, amphibian,
insect, reptilian, fish, mammalian, rodent, equine, ovine, goat,
sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial
DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single,
double or triple stranded, hybridized, and the like or any
combination thereof. Antibody producing cells can also be obtained
from the peripheral blood or, preferably the spleen or lymph nodes,
of humans or other suitable animals that have been immunized with
the antigen of interest. Any other suitable host cell can also be
used for expressing-heterologous or endogenous nucleic acid
encoding an antibody, specified fragment or variant thereof, of the
present invention. The fused cells (hybridomas) or recombinant
cells can be isolated using selective culture conditions or other
suitable known methods, and cloned by limiting dilution or cell
sorting, or other known methods.
Other suitable methods of producing or isolating antibodies of the
requisite specificity can be used, including, but not limited to,
methods that select recombinant antibody from a peptide or protein
library (e.g., but not limited to, a bacteriophage, ribosome,
oligonucleotide, cDNA, or the like, display library; e.g., as
available from various commercial vendors such as MorphoSys
(Martinsreid/Planegg, Del.), BioInvent (Lund, Sweden), Affitech
(Oslo, Norway) using methods known in the art. Art known methods
are described in the patent literature some of which include U.S.
Pat. Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483;
5,824,514; 5,976,862. Alternative methods rely upon immunization of
transgenic animals (e.g., SCID mice) (Nguyen et al., 1977; Sandhu
et al., 1996); Eren et al., 1998), that are capable of producing a
repertoire of human antibodies, as known in the art and/or as
described herein. Such techniques, include, but are not limited to,
ribosome display (Wanes et al., 1997; Hanes et al., 1998); single
cell antibody producing technologies (e.g., selected lymphocyte
antibody method ("SLAM") (U.S. Pat. No. 5,627,052, Wen et al.,
1987; Babcook et al., 1996); gel microdroplet and flow cytometry
(Powell et al., 1990; Gray et al., 1995; Kenny et al., 1995);
B-cell selection (Steenbakkers et al., 1994).
The terms "polyclonal antibody" or "polyclonal antibody
composition" as used herein refer to a preparation of antibodies
that are derived from different B-cell lines. They are a mixture of
immunoglobulin molecules secreted against a specific antigen, each
recognizing a different epitope.
The term "mouse antibody" as used herein, is intended to include
antibodies having variable and constant regions derived from mouse
germline immunoglobulin sequences.
As used herein, chimeric antibodies are antibodies whose light and
heavy chain genes have been constructed, typically by genetic
engineering, from antibody variable and constant region genes
belonging to different species.
In one embodiment, the anti-OX40L antibody or antigen binding
fragment thereof is a neutralizing antibody or antigen-binding
fragment thereof. The term "neutralizing" in the context of an
OX40L neutralizing antibody refers to an antibody that may do one
or more of: interfere with the OX40/OX40L interaction; reduce the
concentration of OX40/OX40L interacted species in a subject or a
cell; prevent the OX40/OX40L interaction in a subject or a cell;
and/or reduce the biological function of OX40L, which may include
one or more of: inhibiting the proliferation and the production of
TNF-a, promoting IL-10 production by CD4.sup.+ T cells, suppressing
the activation of inflammatory T cells, and/or converting the
differentiation of inflammatory T cells into regulatory T
cells.
In further embodiments, the antibody comprises a modification and
is an "antibody derivative." The term "antibody derivative"
includes post-translational modification to linear polypeptide
sequence of the antibody or fragment. For example, U.S. Pat. No.
6,602,684 B1 describes a method for the generation of modified
glycol-forms of antibodies, including whole antibody molecules,
antibody fragments, or fusion proteins that include a region
equivalent to the Fc region of an immunoglobulin, having enhanced
Fe-mediated cellular toxicity, and glycoproteins so generated.
The antibodies of the invention also include derivatives that are
modified by the covalent attachment of any type of molecule to the
antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. Antibody
derivatives include, but are not limited to, antibodies that have
been modified by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Additionally, the
derivatives may contain one or more non-classical amino acids.
Antibody derivatives of the present invention can also be prepared
by delivering a polynucleotide encoding an antibody of this
invention to a suitable host such as to provide transgenic animals
or mammals, such as goats, cows, horses, sheep, and the like, that
produce such antibodies in their milk. These methods are known in
the art and are described for example in U.S. Pat. Nos. 5,827,690;
5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and
5,304,489.
Antibody derivatives also can be prepared by delivering a
polynucleotide of this invention to provide transgenic plants and
cultured plant cells (e.g., but not limited to tobacco, maize, and
duckweed) that produce such antibodies, specified portions or
variants in the plant parts or in cells cultured therefrom.
Antibody derivatives have also been produced in large amounts from
transgenic plant seeds including antibody fragments, such as single
chain antibodies (scFv's), including tobacco seeds and potato
tubers. See, e.g., Conrad et al., 1998 and references cited
therein. Thus, antibodies can also be produced using transgenic
plants, according to know methods.
Antibody derivatives also can be produced, for example, by adding
exogenous sequences to modify immunogenicity or reduce, enhance or
modify binding, affinity, on-rate, off-rate, avidity, specificity,
half-life, or any other suitable characteristic. Generally part or
all of the non-human or human CDR sequences are maintained while
the non-human sequences of the variable and constant regions are
replaced with human or other amino acids.
The term "variable region" refers to a portion of the antibody that
gives the antibody its specificity for binding antigen. The
variable region is typically located at the ends of the heavy and
light chains. Variable loops of .beta.-strands, three each on the
light (VL) and heavy (VH) chains are responsible for binding to the
antigen. These loops are referred to as the "complementarity
determining regions" (CDRs).
In general, the CDR residues are directly and most substantially
involved in influencing antigen binding. Humanization or
engineering of antibodies can be performed using any known method
such as, but not limited to, those described in U.S. Pat. Nos.
5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192;
5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762;
5,530,101; 5,585,089; 5,225,539; and 4,816,567.
The term "constant region" refers to a portion of the antibody that
is identical in all antibodies of the same isotype. The constant
region differs in antibodies of different isotypes.
As used herein, the term "humanized antibody" or "humanized
immunoglobulin" refers to a human/non-human chimeric antibody that
contains a minimal sequence derived from non-human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which residues from a variable region of
the recipient are replaced by residues from a variable region of a
non-human species (donor antibody) such as mouse, rat, rabbit, or
non-human primate having the desired specificity, affinity and
capacity. Humanized antibodies may comprise residues that are not
found in the recipient antibody or in the donor antibody. The
humanized antibody can optionally also comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin, a non-human antibody containing one or more
amino acids in a framework region, a constant region or a CDR, that
have been substituted with a correspondingly positioned amino acid
from a human antibody. In general, humanized antibodies are
expected to produce a reduced immune response in a human host, as
compared to a non-humanized version of the same antibody. The
humanized antibodies may have conservative amino acid substitutions
which have substantially no effect on antigen binding or other
antibody functions. Conservative substitutions groupings include:
glycine-alanine, valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, serine-threonine and
asparagine-glutamine.
Chimeric, humanized or primatized antibodies of the present
invention can be prepared based on the sequence of a reference
monoclonal antibody prepared using standard molecular biology
techniques. DNA encoding the heavy and light chain immunoglobulins
can be obtained from the hybridoma of interest and engineered to
contain non-reference (e.g., human) immunoglobulin sequences using
standard molecular biology techniques. For example, to create a
chimeric antibody, the murine variable regions can be linked to
human constant regions using methods known in the art (U.S. Pat.
No. 4,816,567). To create a humanized antibody, the murine CDR
regions can be inserted into a human framework using methods known
in the art (U.S. Pat. No. 5,225,539 and U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,762 and 6,180,370). Similarly, to create a
primatized antibody the murine CDR regions can be inserted into a
primate framework using methods known in the art (WO 93/02108 and
WO 99/55369).
Techniques for making partially to fully human antibodies are known
in the art and any such techniques can be used. According to one
embodiment, fully human antibody sequences are made in a transgenic
mouse which has been engineered to express human heavy and light
chain antibody genes. Multiple strains of such transgenic mice have
been made which can produce different classes of antibodies. B
cells from transgenic mice which are producing a desirable antibody
can be fused to make hybridoma cell lines for continuous production
of the desired antibody. (See for example, Russel et al., 2000;
Gallo et al., 2000; Green, 1999; Yang et al., 1999A; Yang, 1999B;
Jakobovits, 1998; Green and Jakobovits, 1998; Jakobovits, 1998;
Tsuda et al., 1997; Sherman-Gold, 1997; Mendez et al., 1997;
Jakobovits, 1996; Jakobovits, 1995; Mendez et al, 1995; Jakobovits,
1994; Arbones et al., 1994; Jakobovits, 1993; Jakobovits et al.,
1993; U.S. Pat. No. 6,075,181.)
The antibodies of this invention also can be modified to create
chimeric antibodies. Chimeric antibodies are those in which the
various domains of the antibodies' heavy and light chains are coded
for by DNA from more than one species. See, e.g., U.S. Pat. No.
4,816,567.
Alternatively, the antibodies of this invention can also be
modified to create veneered antibodies. Veneered antibodies are
those in which the exterior amino acid residues of the antibody of
one species are judiciously replaced or "veneered" with those of a
second species so that the antibodies of the first species will not
be immunogenic in the second species thereby reducing the
immunogenicity of the antibody. Since the antigenicity of a protein
is primarily dependent on the nature of its surface, the
immunogenicity of an antibody could be reduced by replacing the
exposed residues which differ from those usually found in another
mammalian species antibodies. This judicious replacement of
exterior residues should have little, or no, effect on the interior
domains, or on the interdomain contacts. Thus, ligand binding
properties should be unaffected as a consequence of alterations
which are limited to the variable region framework residues. The
process is referred to as "veneering" since only the outer surface
or skin of the antibody is altered, the supporting residues remain
undisturbed.
The procedure for "veneering" makes use of the available sequence
data for human antibody variable domains compiled by Kabat et al.
(1987) Sequences of Proteins of Immunological interest, 4th ed.,
Bethesda, Md., National Institutes of Health, updates to this
database, and other accessible U.S. and foreign databases (both
nucleic acid and protein). Non-limiting examples of the methods
used to generate veneered antibodies include EP 519596; U.S. Pat.
No. 6,797,492; and described in Padlan et al., 1991.
The term "antibody derivative" also includes "diabodies" which are
small antibody fragments with two antigen-binding sites, wherein
fragments comprise a heavy chain variable domain (VH) connected to
a light chain variable domain (VL) in the same polypeptide chain.
(See for example, EP 404,097; WO 93/11161; and Hollinger, et al.,
1993) By using a linker that is too short to allow pairing between
the two domains on the same chain, the domains are forced to pair
with the complementary domains of another chain and create two
antigen-binding sites. (See also, U.S. Pat. No. 6,632,926 to Chen
et al, which discloses antibody variants that have one or more
amino acids inserted into a hypervariable region of the parent
antibody and a binding affinity for a target antigen which is at
least about two fold stronger than the binding affinity of the
parent antibody for the antigen).
The term "antibody derivative" further includes engineered antibody
molecules, fragments and single domains such as scFv, dAbs,
nanobodies, minibodies, Unibodies, and Affibodies (Holliger &
Hudson, 2005; U.S. Patent Publication US 2006/0211088; PCT
Publication WO2007/059782; U.S. Pat. No. 5,831,012).
The term "antibody derivative" further includes "linear
antibodies". The procedure for making linear antibodies is known in
the art and described in Zapata et al., 1995. Briefly, these
antibodies comprise a pair of tandem Ed segments
(V.sub.H-C.sub.H1-VH-C.sub.H1) which form a pair of antigen binding
regions. Linear antibodies can be bispecific or monospecific.
The antibodies of this invention can be recovered and purified from
recombinant cell cultures by known methods including, but not
limited to, protein A purification, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be used for
purification.
In certain embodiments, the antibodies of the invention are
recombinant antibodies. A recombinant antibody differs from an
endogenously-produced antibody. For example, recombinant antibodies
differ with respect to their glycosylation status (see, for
example, Jefferis, R. "Glycolsylation of Recombinant Antibody
Therapeutics" Biotechnol. Prog. 2005, 21:11-16 which is herein
incorporated by reference).
In some embodiments, the antibody is an engineered antibody. For
example, the Fc region may be engineered to increase binding to
Fc.gamma. receptors of effector cells. This may involve modifying
antibody glycosylation patterns or mutating amino acids in the Fc
region. Glycoengineering may be performed by methods known in the
art such as POTELLIGENT and Glycart. Methods for amino acid
engineering are also known and used in the art (e.g. Xmab approach
by Xencor). Amino acid changes to the Fc region can improve
antibody-dependent cell-mediated cytotoxicity and complement
dependent cytotoxicity by way of improved binding to effector
cells, but may also allow for an extended half-life. See Evans and
Syed, Nature Reviews, 2014, 13:413-414, for further examples.
Antibodies of the disclosure may be mono-, bi-, or multi-specific.
Bi and multi-specific antibodies are antibodies that recognize two
or multiple antigenic targets. There are some multi-specific
antibody platforms commercially in use, which include the "BiTE"
(bispecific T cell engager) platform, and the DART platform.
Multispecific antibodies, engineered antibodies and various other
platforms are described in Evans and Syed, "Next-generation
antibodies," Nature Reviews, 2014, 13:413-414, which is hereby
incorporated by reference in its entirety.
If an antibody being tested binds with protein or polypeptide, then
the antibody being tested and the antibodies provided by this
invention are equivalent. In one embodiment, an equivalent is one
that binds OX40L and provides the same neutralizing activity and/or
cell response (i.e. inhibit the differentiation of IL-10 low/TNF-a
high producing inflammatory Th2 primed by TSLP-mDCs and/or inhibit
the proliferation and the production of TNF-1 and promote IL-10 by
CD4+ T cells).
It also is possible to determine without undue experimentation,
whether an antibody has the same specificity as the antibody of
this invention by determining whether the antibody being tested
prevents an antibody of this invention from binding the protein or
polypeptide with which the antibody is normally reactive. If the
antibody being tested competes with the antibody of the invention
as shown by a decrease in binding by the monoclonal antibody of
this invention, then it is likely that the two antibodies bind to
the same or a closely related epitope. Alternatively, one can
pre-incubate the antibody of this invention with a protein with
which it is normally reactive, and determine if the antibody being
tested is inhibited in its ability to bind the antigen. If the
antibody being tested is inhibited then, in all likelihood, it has
the same, or a closely related, epitopic specificity as the
antibody of this invention.
The term "antibody" also is intended to include antibodies of all
immunoglobulin isotypes and subclasses unless specified otherwise.
An isotype refers to the genetic variations or differences in the
constant regions of the heavy and light chains of an antibody. In
humans, there are five heavy chain isotypes: IgA, IgD, IgG, IgE,
and IgM and two light chain isotypes: kappa and lambda. The IgG
class is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4 in
humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. They share more
than 95% homology in the amino acid sequences of the Fc regions but
show major differences in the amino acid composition and structure
of the hinge region. Particular isotypes of a monoclonal antibody
can be prepared either directly by selecting from an initial
fusion, or prepared secondarily, from a parental hybridoma
secreting a monoclonal antibody of different isotype by using the
sib selection technique to isolate class switch variants using the
procedure described in Steplewski et al., 1985; Spira et al, 1984).
Alternatively, recombinant DNA techniques may be used.
The isolation of other monoclonal antibodies with the specificity
of the monoclonal antibodies described herein can also be
accomplished by one of ordinary skill in the art by producing
anti-idiotypic antibodies (Herlyn et al., 1986). An anti-idiotypic
antibody is an antibody which recognizes unique determinants
present on the monoclonal antibody of interest.
In some aspects of this invention, it will be useful to detectably
or therapeutically label the antibody. Methods for conjugating
antibodies to these agents are known in the art. For the purpose of
illustration only, antibodies can be labeled with a detectable
moiety such as a radioactive atom, a chromophore, a fluorophore, or
the like. Such labeled antibodies can be used for diagnostic
techniques, either in vivo, or in an isolated test sample.
As used herein, the term "label" intends a directly or indirectly
detectable compound or composition that is conjugated directly or
indirectly to the composition to be detected, e.g., polynucleotide
or protein such as an antibody so as to generate a "labeled"
composition. The term also includes sequences conjugated to the
polynucleotide that will provide a signal upon expression of the
inserted sequences, such as green fluorescent protein (GFP) and the
like. The label may be detectable by itself (e.g. radioisotope
labels or fluorescent labels) or, in the case of an enzymatic
label, may catalyze chemical alteration of a substrate compound or
composition which is detectable. The labels can be suitable for
small scale detection or more suitable for high-throughput
screening. As such, suitable labels include, but are not limited to
radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and
proteins, including enzymes. The label may be simply detected or it
may be quantified. A response that is simply detected generally
comprises a response whose existence merely is confirmed, whereas a
response that is quantified generally comprises a response having a
quantifiable (e.g., numerically reportable) value such as an
intensity, polarization, and/or other property. In luminescence or
fluorescence assays, the detectable response may be generated
directly using a luminophore or fluorophore associated with an
assay component actually involved in binding, or indirectly using a
luminophore or fluorophore associated with another (e.g., reporter
or indicator) component.
Examples of luminescent labels that produce signals include, but
are not limited to bioluminescence and chemiluminescence.
Detectable luminescence response generally comprises a change in,
or an occurrence of, a luminescence signal. Suitable methods and
luminophores for luminescently labeling assay components are known
in the art and described for example in Haugland, Richard P. (1996)
Handbook of Fluorescent Probes and Research Chemicals (6.sup.th
ed.). Examples of luminescent probes include, but are not limited
to, aequorin and luciferases.
Examples of suitable fluorescent labels include, but are not
limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin,
erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,
stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other
suitable optical dyes are described in the Haugland, Richard P.
(1996) Handbook of Fluorescent Probes and Research Chemicals
(6.sup.th ed.).
In another aspect, the fluorescent label is functionalized to
facilitate covalent attachment to a cellular component present in
or on the surface of the cell or tissue such as a cell surface
marker. Suitable functional groups, including, but not are limited
to, isothiocyanate groups, amino groups, haloacetyl groups,
maleimides, succinimidyl esters, and sulfonyl halides, all of which
may be used to attach the fluorescent label to a second molecule.
The choice of the functional group of the fluorescent label will
depend on the site of attachment to either a linker, the agent, the
marker, or the second labeling agent.
Attachment of the fluorescent label may be either directly to the
cellular component or compound or alternatively, can by via a
linker. Suitable binding pairs for use in indirectly linking the
fluorescent label to the intermediate include, but are not limited
to, antigens/antibodies, e.g., rhodamine/anti-rhodamine,
biotin/avidin and biotin/strepavidin.
The coupling of antibodies to low molecular weight haptens can
increase the sensitivity of the antibody in an assay. The haptens
can then be specifically detected by means of a second reaction.
For example, it is common to use haptens such as biotin, which
reacts avidin, or dinitrophenol, pyridoxal, and fluorescein, which
can react with specific anti-hapten antibodies. See, Harlow and
Lane (1988) supra.
The variable region of the antibodies of the present invention can
be modified by mutating amino acid residues within the VH and/or VL
CDR 1, CDR 2 and/or CDR 3 regions to improve one or more binding
properties (e.g., affinity) of the antibody. Mutations may be
introduced by site-directed mutagenesis or PCR-mediated mutagenesis
and the effect on antibody binding, or other functional property of
interest, can be evaluated in appropriate in vitro or in vivo
assays. Preferably conservative modifications are introduced and
typically no more than one, two, three, four or five residues
within a CDR region are altered. The mutations may be amino acid
substitutions, additions or deletions.
Framework modifications can be made to the antibodies to decrease
immunogenicity, for example, by "backmutating" one or more
framework residues to the corresponding germline sequence.
In addition, the antibodies of the invention may be engineered to
include modifications within the Fc region to alter one or more
functional properties of the antibody, such as serum half-fife,
complement fixation, Fc receptor binding, and/or antigen-dependent
cellular cytotoxicity. Such modifications include, but are not
limited to, alterations of the number of cysteine residues in the
hinge region to facilitate assembly of the light and heavy chains
or to increase or decrease the stability of the antibody (U.S. Pat.
No. 5,677,425) and amino acid mutations in the Fc hinge region to
decrease die biological half life of the antibody (U.S. Pat. No.
6,165,745).
Additionally, the antibodies of the invention may be chemically
modified. Glycosylation of an antibody can be altered, for example,
by modifying one or more sites of glycosylation within the antibody
sequence to increase the affinity of the antibody for antigen (U.S.
Pat. Nos. 5,714,350 and 6,350,861). Alternatively, to increase
antibody-dependent cell-mediated cytotoxicity, a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures can be obtained by
expressing the antibody in a host cell.sub.--with altered
glycosylation mechanism (Shields, et al., 2002; Umana et al.,
1999).
The antibodies of the invention can be pegylated to increase
biological half-life by reacting the antibody or fragment thereof
with polyethylene glycol (PEG) or a reactive ester or aldehyde
derivative of PEG, under conditions in which one or more PEG groups
become attached to the antibody or antibody fragment. Antibody
pegylation may be carried out by an acylation reaction or an
alkylation reaction with a reactive PEG molecule (or an analogous
reactive watersoluble polymer). As used herein, the term
"polyethylene glycol" is intended to encompass any of the forms of
PEG that have been used to derivatize other proteins, such as mono
(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. The antibody to be pegylated can be an
aglycosylated antibody. Methods for pegylating proteins are known
in the art and can be applied to the antibodies of the invention
(EP 0 154 316 and EP 0 401 384).
Additionally, antibodies may be chemically modified by conjugating
or fusing the antigen-binding region of the antibody to serum
protein, such as human serum albumin, to increase half-life of the
resulting molecule. Such approach is for example described in EP
0322094 and EP 0 486 525.
The antibodies or fragments thereof of the present invention may be
conjugated to a diagnostic agent and used diagnostically, for
example, to monitor the development or progression of a disease and
determine the efficacy of a given treatment regimen. Examples of
diagnostic agents include enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody or fragment thereof, or indirectly,
through a linker using techniques known in the art. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase. Examples
of suitable prosthetic group complexes include streptavidin/biotin
and avidin/biotin. Examples of suitable fluorescent materials
include umbelliferone, fluorescein, fluorescein isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin. An example of a luminescent material includes
luminol. Examples of bioluminescent materials include luciferase,
luciferin, and aequorin. Examples of suitable radioactive material
include .sup.125I, .sup.131I, Indium-111, Lutetium-171,
Bismuth-212, Bismuth-213, Astatine-211, Copper-62, Copper-64,
Copper-67, Yttrium-90, Iodine-125, Iodine-131, Phosphorus-32,
Phosphorus-33, Scandium-47, Silver-111, Gallium-67,
Praseodymium-142, Samarium-153, Terbium-161, Dysprosium-166,
Holmium-166, Rhenium-186, Ithenium-188, Rhenium-189, Lead-212,
Radium-223, Actinium-225, Iron-59, Selenium-75, Arsenic-77,
Strontium-89, Molybdenum-99, Rhodium-1105, Palladium-109,
Praseodymium-143, Promethium-149, Erbium-169, Iridium-194,
Gold-198, Gold-199, and Lead-211. Monoclonal antibodies may be
indirectly conjugated with radiometal ions through the use of
bifunctional chelating agents that are covalently linked to the
antibodies. Chelating agents may be attached through amities
(Meares et al., 1984); sulfhydral groups (Koyama 1994) of amino
acid residues and carbohydrate groups (Rodwell et al., 1986; Quadri
et al., 1993).
Additional suitable conjugated molecules include ribonuclease
(RNase), DNase I, an antisense nucleic acid, an inhibitory RNA
molecule such as a siRNA molecule, an immunostimulatory nucleic
acid, aptamers, ribozymes, triplex forming molecules, and external
guide sequences. Aptamers are small nucleic acids ranging from
15-50 bases in length that fold into defined secondary and tertiary
structures, such as stern-loops or G-quartets, and can bind small
molecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline
(U.S. Pat. No. 5,580,737), as well as large molecules, such as
reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S.
Pat. No. 5,543,293). Ribozymes are nucleic acid molecules that are
capable of catalyzing a chemical reaction, either intramolecularly
or intermolecularly. Ribozymes typically cleave nucleic acid
substrates through recognition and binding of the target substrate
with subsequent cleavage. Triplex forming function nucleic acid
molecules can interact with double-stranded or single-stranded
nucleic acid by forming a triplex, in which three strands of DNA
form a complex dependant on both Watson-Crick and Hoogsteen
base-pairing. Triplex molecules can bind target regions with high
affinity and specificity.
The functional nucleic acid molecules may act as effectors,
inhibitors, modulators, and stimulators of a specific activity
possessed by a target molecule, or the functional nucleic acid
molecules may possess a de novo activity independent of any other
molecules.
The conjugated agents can be linked to the antibody directly or
indirectly, using any of a large number of available methods. For
example, an agent can be attached at the hinge region of the
reduced antibody component via disulfide bond formation, using
cross-linkers such as N-succinyl 3-(2-pyridyldithio)proprionate
(SPDP), or via a carbohydrate moiety in the Fc region of the
antibody (Yu et al., 1994; Upeslacis et al., 1995; Price,
1995).
Techniques for conjugating agents to antibodies are well known
(Amon et al., 1985; Hellstrom et al., 1987; Thorpe, 1985; Baldwin
et al., 1985; Thorpe et al., 1982),
The antibodies of the invention or antigen-binding regions thereof
can be linked to another functional molecule such as another
antibody or ligand for a receptor to generate a bi-specific or
multi-specific molecule that binds to at least two or more
different binding sites or target molecules. Linking of the
antibody to one or more other binding molecules, such as another
antibody, antibody fragment, peptide or binding mimetic, can be
done, for example, by chemical coupling, genetic fusion, or
noncovalent association. Multi-specific molecules can further
include a third binding specificity, in addition to the first and
second target epitope.
Bi-specific and multi-specific molecules can be prepared using
methods known in the art. For example, each binding unit of the
hi-specific molecule can be generated separately and then
conjugated to one another. When the binding molecules are proteins
or peptides, a variety of coupling or cross-linking agents can be
used for covalent conjugation. Examples of cross-linking agents
include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitroberizoic acid) (DTNB), o-phenylenedimaleimide
(oRDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-I-carboxylate
(sulfo-SMCC) (Karpovsky et al., 1984; Liu et al., 1985). When the
binding molecules are antibodies, they can be conjugated by
sulfhydryl bonding of the C-terminus hinge regions of the two heavy
chains.
The antibodies or fragments thereof of the present invention may be
linked to a moiety that is toxic to a cell to which the antibody is
bound to form "depleting" antibodies. These antibodies are
particularly useful in applications where it is desired to deplete
an NK cell.
The antibodies of the invention may also be attached to solid
supports, which are particularly useful for immunoassays or
purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
The antibodies also can be bound to many different carriers. Thus,
this invention also provides compositions containing the antibodies
and another substance, active or inert. Examples of well-known
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylase, natural and modified cellulose,
polyacrylamide, agarose, and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding monoclonal antibodies, or will be able to ascertain such,
using routine experimentation.
B. Other OX40L Inhibitors
OX40L inhibitors include polynucleotides that decreases the
biological activity of the OX40L gene and/or protein and can be,
for example, a miRNA, a siRNA, a shRNA, a dsRNA or an antisense RNA
directed to OX40L DNA or mRNA, or a polynucleotide encoding the
miRNA, siRNA, shRNA, dsRNA or antisense RNA, or a vector comprising
the polynucleotide.
"Short interfering RNAs" (siRNA) refer to double-stranded RNA
molecules (dsRNA), generally, from about 10 to about 30 nucleotides
in length that are capable of mediating RNA interference (RNAi).
"RNA interference" (RNAi) refers to sequence-specific or gene
specific suppression of gene expression (protein synthesis) that is
mediated by short interfering RNA (siRNA). As used herein, the term
siRNA includes short hairpin RNAs (shRNAs). A siRNA directed to a
gene or the mRNA of a gene may be a siRNA that recognizes the mRNA
of the gene and directs a RNA-induced silencing complex (RISC) to
the mRNA, leading to degradation of the mRNA. A siRNA directed to a
gene or the mRNA of a gene may also be a siRNA that recognizes the
mRNA and inhibits translation of the mRNA. A siRNA may be
chemically modified to increase its stability and safety. See, e.g.
Dykxhoom & Lieberman, 2006 and U.S. Patent Application
Publication No.: 2008/0249055.
"Double stranded RNAs" (dsRNA) refer to double stranded RNA
molecules that may be of any length and may be cleaved
intracellularly into smaller RNA molecules, such as siRNA. In cells
that have a competent interferon response, longer dsRNA, such as
those longer than about 30 base pair in length, may trigger the
interferon response. In other cells that do not have a competent
interferon response, dsRNA may be used to trigger specific
RNAi.
"MicroRNAs" (miRNA) refer to single-stranded RNA molecules of 21-23
nucleotides in length, which regulate gene expression. miRNAs are
encoded by genes from whose DNA they are transcribed but miRNAs are
not translated into protein (non-coding RNA); instead each primary
transcript (a pri-miRNA) is processed into a short stem-loop
structure called a pre-miRNA and finally into a functional miRNA.
Mature miRNA molecules are partially complementary to one or more
messenger RNA (mRNA) molecules, and their main function is to
down-regulate gene expression.
siRNA, dsRNA, and miRNA to inhibit gene expression can be designed
following procedures known in the art. See, e.g., Dykxhoorn &
Lieberman, 2006; Dykxhoorn et al., 2006; Aagaard & Rossi, 2007;
de Fougerolles et al., 2007; Krueger et al., 2007; U.S. Patent
Application Publication No.: 2008/0188430; and U.S. Patent
Application Publication No.: 2008/0249055.
Delivery of siRNA, dsRNA or miRNA to a cell can be made with
methods known in the art. See, e.g., Dykxhoorn & Lieberman,
2006; Dykxhoorn et al., 2006; Aagaard & Rossi, 2007; de
Fougerolles et al., 2007; Krueger et al., 2007; U.S. Patent
Application Publication No.: 2008/0188430; and U.S. Patent
Application Publication No.: 2008/0249055. "Antisense"
oligonucleotides have nucleotide sequences complementary to the
protein coding or "sense" sequence. Antisense RNA sequences
function as regulators of gene expression by hybridizing to
complementary mRNA sequences and arresting translation (Mizuno et
al., 1984; Heywood et al., 1986). An antisense polynucleotide
comprising the entire sequence of the target transcript or any part
thereof can be synthesized with methods known in the art. See e.g.,
Ferretti et al., 1986. The antisense polynucleotide can be placed
into vector constructs, and effectively introduced into cells to
inhibit gene expression (Izant et al., 1984). Generally, to assure
specific hybridization, the antisense sequence is substantially
complementary to the target sequence. In certain embodiments, the
antisense sequence is exactly complementary to the target sequence.
The antisense polynucleotides may also include, however, nucleotide
substitutions, additions, deletions, transitions, transpositions,
or modifications, or other nucleic acid sequences or non-nucleic
acid moieties so long as specific binding to the relevant target
sequence corresponding to the gene is retained as a functional
property of the polynucleotide.
The antisense nucleic acids (DNA, RNA, modified, analogues, and the
like) can be made using any suitable method for producing a nucleic
acid, such as the chemical synthesis and recombinant methods
disclosed herein and known to one of skill in the art. In one
embodiment, for example, antisense RNA molecules of the invention
may be prepared by de novo chemical synthesis or by cloning. For
example, an antisense RNA can be made by inserting (ligating) a
gene sequence in reverse orientation operably linked to a promoter
in a vector (e.g., plasmid). Provided that the promoter and,
preferably termination and polyadenylation signals, are properly
positioned, the strand of the inserted sequence corresponding to
the noncoding strand will be transcribed and act as an antisense
oligonucleotide of the invention.
It will be appreciated that the oligonucleotides can be made using
nonstandard bases (e.g., other than adenine, cytidine, guanine,
thymine, and uridine) or nonstandard backbone structures to
provides desirable properties (e.g., increased nuclease-resistance,
tighter-binding, stability or a desired T.sub.m). Techniques for
rendering oligonucleotides nuclease-resistant include those
described in PCT Publication WO 94/12633. A wide variety of useful
modified oligonucleotides may be produced, including
oligonucleotides having a peptide-nucleic acid (PNA) backbone
(Nielsen et al., 1991) or incorporating 2'-O-methyl
ribonucleotides, phosphorothioate nucleotides, methyl phosphonate
nucleotides, phosphotriester nucleotides, phosphorothioate
nucleotides, phosphoramidates. Another example of the modification
is replacement of a non-bridging phosphoryl oxygen atom with a
sulfur atom which increases resistance to nuclease digestion.
Increased antisense polynucleotide stability can also be achieved
using molecules with 2-methyoxyethyl substituted backbones. See
e.g., U.S. Pat. Nos. 6,451,991 and 6,900,187.
In another embodiment, ribozymes can be used (see, e.g., Cech,
1995; and Edgington, 1992; Hu et al., PCT Publication WO 94/03596).
A ribonucleic acid enzyme ("ribozymes", "RNA enzyme", or "catalytic
RNA") is an RNA molecule that catalyzes a chemical reaction. Many
natural ribozymes catalyze either the hydrolysis of one of their
own phosphodiester bonds, or the hydrolysis of bonds in other RNAs,
but they have also been found to catalyze the aminotransferase
activity of the ribosome. Methods of making and using ribozymes can
be found in e.g., U.S. Patent Application Publication No.
2006/0178326.
"Triplex ribozymes" configurations allow for increased target
cleavage relative to conventionally expressed ribozymes. Examples
of triplex ribozymes include hairpin ribozymes and hammerhead
ribozymes. Methods of making and using triplex ribozymes are found
in, e.g., Aguino-Jarguin et al., 2008 and U.S. Patent Application
Publication No. 2005/0260163.
Proteins have been described that have the ability to translocate
desired nucleic acids across a cell membrane. Typically, such
proteins have amphiphilic or hydrophobic subsequences that have the
ability to act as membrane-translocating carriers. For example,
homeodomain proteins have the ability to translocate across cell
membranes. The shortest internalizable peptide of a homeodomain
protein, Antennapedia, was found to be the third helix of the
protein, from amino acid position 43 to 58 (see, e.g., Prochiantz,
1996). Another subsequence, the h (hydrophobic) domain of signal
peptides, was found to have similar cell membrane translocation
characteristics (see, e.g., Lin et al., 1995). Such subsequences
can be used to translocate oligonucleotides across a cell membrane.
Oligonucleotides can be conveniently derivatized with such
sequences. For example, a linker can be used to link the
oligonucleotides and the translocation sequence. Any suitable
linker can be used, e.g., a peptide linker or any other suitable
chemical linker.
This disclosure features methods and compositions for decreasing
the biological activity of OX40L in a cell. The OX40L inhibitors
described herein may be expressed in cells using expression
vectors, viral vectors, and other techniques known in the art for
transferring genetic material to the cells of a patient with
resulting therapeutic benefit to the patient. In some embodiments,
polypeptides or polynucleotides encoding the polypeptides (e.g.
antibodies and antibody fragments) described herein may be
delivered to humans or to host cells.
In some embodiments, expression vectors encoding the OX40L
inhibitor polynucleotide of interest is administered directly to
the patient. The vectors are taken up by the target cells (e.g.,
neurons or pluripotent stem cells) and the polynucleotide is
expressed. Recent reviews discussing methods and compositions for
use in gene therapy include Eck et al., in Goodman & Gilman's
The Pharmacological Basis of Therapeutics, 1996; Wilson, 1997;
Wivel et al., 1998; Romano et al., 2000). U.S. Pat. No. 6,080,728
also provides a discussion of a wide variety of gene delivery
methods and compositions.
Adenoviruses are able to transfect a wide variety of cell types,
including non-dividing cells. There are more than 50 serotypes of
adenoviruses that are known in the art, but the most commonly used
serotypes for gene therapy are type 2 and type 5. Typically, these
viruses are replication-defective; and genetically-modified to
prevent unintended spread of the virus. This is normally achieved
through the deletion of the E1 region, deletion of the E1 region
along with deletion of either the E2 or E4 region, or deletion of
the entire adenovirus genome except the cis-acting inverted
terminal repeats and a packaging signal (Gardlik et al., 2005).
Retroviruses are also useful as vectors and usually (with the
exception of lentiviruses) are not capable of transfecting
non-dividing cells. Accordingly, any appropriate type of retrovirus
that is known in the art may be used, including, but not limited
to, HIV, SIV, FIV, EIAV, and Moloney Murine Leukaemia Virus
(MoMLV). Typically, therapeutically useful retroviruses including
deletions of the gag, pol, or env genes.
In another aspect, the invention features the methods of inhibiting
OX40L with a lentiviral vector that expresses an OX40L inhibiting
polynucleotide in a patient. Lentiviruses are a type of
retroviruses with the ability to infect both proliferating and
quiescent cells. An exemplary lentivirus vector for use in gene
therapy is the HIV-1 lentivirus. Previously constructed genetic
modifications of lentiviruses include the deletion of all protein
encoding genes except those of the gag, pol, and rev genes
(Moreau-Gaudry et al., 2001)
Exemplary non-viral vectors for delivering nucleic acids include
naked DNA; DNA complexed with cationic lipids, alone or in
combination with cationic polymers; anionic and cationic liposomes;
DNA-protein complexes and particles comprising DNA condensed with
cationic polymers such as heterogeneous polylysine, defined-length
oligopeptides, and polyethylene imine, in some cases contained in
liposomes; and the use of ternary complexes comprising a virus and
polylysine-DNA. In vivo DNA-mediated gene transfer into a variety
of different target sites has been studied extensively. Naked DNA
may be administered using an injection, a gene gun, or
electroporation. Naked DNA can provide long-term expression in
muscle. See Wolff et al., 1992; Wolff et al., 1990. DNA-mediated
gene transfer has also been characterized in liver, heart, lung,
brain and endothelial cells. See Zhu et al., 1993; Nabel et al.,
1989. DNA for gene transfer also may be used in association with
various cationic lipids, polycations and other conjugating
substances. See Przybylska et al., 2004; Svahn et al., 2004.
Methods of delivering nucleic acids using cationic liposomes are
also well known in the art. Exemplary cationic liposomes for use in
this invention are DOTMA, DOPE, DOSPA, DOTAP, DC-Chol, Lipid
GL-67.TM., and EDMPC. These liposomes may be used in vivo or ex
vivo to encapsulate a vector for delivery into target cells (e.g.,
neurons or pluripotent stem cells).
Typically, vectors made in accordance with the principles of this
disclosure will contain regulatory elements that will cause
constitutive expression of the coding sequence. Desirably,
neuron-specific regulatory elements such as neuron-specific
promoters are used in order to limit or eliminate ectopic gene
expression in the event that the vector is incorporated into cells
outside of the target region. Several regulatory elements are well
known in the art to direct neuronal specific gene expression
including, for example, the neural-specific enolase (NSE), and
synapsin-1 promoters (Morelli et al., 1999).
Also provided are polynucleotides encoding substantially homologous
and biologically equivalent polypeptides to the inventive
polypeptides and polypeptide complexes. Substantially homologous
and biologically equivalent intends those having varying degrees of
homology, such as at least 80%, or alternatively, at least 85%, or
alternatively at least 90%, or alternatively, at least 95%, or
alternatively at least 98% homologous as defined above and which
encode polypeptides having the biological activity as described
herein. It should be understood although not always explicitly
stated that embodiments to substantially homologous polypeptides
and polynucleotides are intended for each aspect of this
disclosure, e.g., polypeptides, polynucleotides and antibodies.
The polynucleotides of this disclosure can be replicated using
conventional recombinant techniques. Alternatively, the
polynucleotides can be replicated using PCR technology. PCR is the
subject matter of U.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065;
and 4,683,202 and described in PCR: The Polymerase Chain Reaction
(Mullis et al. eds, Birkhauser Press, Boston (1994)) and references
cited therein. Yet further, one of skill in the art can use the
sequences provided herein and a commercial DNA synthesizer to
replicate the DNA. Accordingly, this disclosure also provides a
process for obtaining the polynucleotides of this disclosure by
providing the linear sequence of the polynucleotide, appropriate
primer molecules, chemicals such as enzymes and instructions for
their replication and chemically replicating or linking the
nucleotides in the proper orientation to obtain the
polynucleotides. In a separate embodiment, these polynucleotides
are further isolated. Still further, one of skill in the art can
operatively link the polynucleotides to regulatory sequences for
their expression in a host cell, described below. The
polynucleotides and regulatory sequences are inserted into the host
cell (prokaryotic or eukaryotic) for replication and amplification.
The DNA so amplified can be isolated from the cell by methods well
known to those of skill in the art. A process for obtaining
polynucleotides by this method is further provided herein as well
as the polynucleotides so obtained.
Also provided are host cells comprising one or more of the
polypeptides or polynucleotides of this disclosure. Yet another
aspect of the disclosure provides an isolated transformed host cell
expressing an isolated polypeptide, an antibody or a biologically
active fragment of the antibody of the disclosure. The isolated
host cells can be a prokaryotic or a eukaryotic cell. In one
aspect, the polypeptides are expressed and can be isolated from the
host cells. In another aspect, the polypeptides are expressed and
secreted. In yet another aspect, the polypeptides are expressed and
present on the cell surface (extracellularly). Suitable cells
containing the inventive polypeptides include prokaryotic and
eukaryotic cells, which include, but are not limited to bacterial
cells, algae cells, yeast cells, insect cells, plant cells, animal
cells, mammalian cells, murine cells, rat cells, sheep cells,
simian cells and human cells. A non-limiting example of algae cells
is red alga Griffithsia sp. from which Griffithsin was isolated
(Toshiyuki et al., 2005). A non-limiting example of plant cells is
a Nicotiana benthamiana leaf cell from which Griffithsin can be
produced in a large scale (O'Keefe, 2009). Examples of bacterial
cells include Escherichia coli (Giomarelli et al. (2006), supra),
Salmonella enteric, Streptococcus gordonii and lactobacillus (Liu
et al., 2007; Rao et al., 2005; Chang et al., 2003; Liu et al.,
2006). The cells can be purchased from a commercial vendor such as
the American Type Culture Collection (ATCC, Rockville Md., USA) or
cultured from an isolate using methods known in the art. Examples
of suitable eukaryotic cells include, but are not limited to 293T
HEK cells, as well as the hamster cell line CHO, BHK-21; the murine
cell lines designated NIH3T3, NSO, C127, the simian cell lines COS,
Vero; and the human cell lines HeLa, PER.C6 (commercially available
from Crucell) U-937 and Hep G2. A non-limiting example of insect
cells include Spodoptera frugiperda. Examples of yeast useful for
expression include, but are not limited to Saccharomyces,
Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or
Pichia. See e.g., U.S. Pat. Nos. 4,812,405; 4,818,700; 4,929,555;
5,736,383; 5,955,349; 5,888,768 and 6,258,559.
The OX40L inhibitor may also be a molecular inhibitor. Methods for
screening various agents that modulate the activity of the protein
are known in the art and are described herein. For the purposes of
this disclosure, a "molecular inhibitor" is intended to include,
but not be limited to a biological or chemical compound such as a
simple or complex organic or inorganic molecule, a peptide, a
protein (e.g. antibody), a polynucleotide (e.g. anti-sense) or a
ribozyme. A vast array of compounds can be synthesized, for example
polymers, such as polypeptides and polynucleotides, and synthetic
organic compounds based on various core structures, and these are
also included in the term "molecular inhibitor." In addition,
various natural sources can provide compounds for screening, such
as plant or animal extracts, and the like. It should be understood,
although not always explicitly stated that the molecular inhibitor
is used alone or in combination with another agent, having the same
or different biological activity as the agents identified by the
inventive screen.
In some embodiments, the OX40L inhibitor is a small molecule
capable of interacting with the OX40L protein. For the purpose of
this invention, "small molecules" are molecules having low
molecular weights (MW) that are, in one embodiment, capable of
binding to a protein of interest thereby altering the function of
the protein. Preferably, the MW of a small molecule is no more than
1,000. Methods for screening small molecules capable of altering
protein function are known in the art. For example, a miniaturized
arrayed assay for detecting small molecule-protein interactions in
cells is discussed by You et al., 1997).
To screen for small molecule inhibitors in vitro, suitable cell
culture or tissue infected with the microbial to be treated are
first provided. The cells are cultured under conditions
(temperature, growth or culture medium and gas (CO.sub.2)) and for
an appropriate amount of time to attain exponential proliferation
without density dependent constraints. It also is desirable to
maintain an additional separate cell culture that is not infected
as a control.
As is apparent to one of skill in the art, suitable cells can be
cultured in micro-titer plates and several small molecule
inhibitors can be assayed at the same time by noting genotypic
changes, phenotypic changes or a reduction in microbial titer. The
small molecule inhibitor can be directly added to the cell culture
or added to culture medium for addition. As is apparent to those
skilled in the art, an "effective" amount must be added which can
be empirically determined.
II. Pharmaceutical Compositions
The present invention includes methods and compositions for
inhibiting OX40L in a subject in need thereof. Administration of
the compositions according to the present invention will typically
be via any common route. This includes, but is not limited to
parenteral, orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal, intranasal, or intravenous injection. In certain
embodiments, a vaccine composition may be inhaled (e.g., U.S. Pat.
No. 6,651,655, which is specifically incorporated by reference).
Additional formulations which are suitable for other modes of
administration include oral formulations. Oral formulations include
such normally employed excipients as, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate and the like. These
compositions take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain about 10% to about 95% of active ingredient, preferably
about 25% to about 70%.
Typically, compositions of the invention are administered in a
manner compatible with the dosage formulation, and in such amount
as will be therapeutically effective and immune modifying. The
quantity to be administered depends on the subject to be treated.
Precise amounts of active ingredient required to be administered
depend on the judgment of the practitioner.
The manner of application may be varied widely. Any of the
conventional methods for administration of an antibody are
applicable. These are believed to include oral application on a
solid physiologically acceptable base or in a physiologically
acceptable dispersion, parenterally, by injection and the like. The
dosage of the pharmaceutical composition will depend on the route
of administration and will vary according to the size and health of
the subject.
In many instances, it will be desirable to have multiple
administrations of at most about or at least about 3, 4, 5, 6, 7,
8, 9, 10 or more. The administrations may range from 2 day to
twelve week intervals, more usually from one to two week intervals.
The course of the administrations may be followed by assays for
alloreactive immune responses and T cell activity.
The phrases "pharmaceutically acceptable" or "pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic, or other untoward reaction when
administered to an animal, or human. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredients, its use in
immunogenic and therapeutic compositions is contemplated.
The pharmaceutical compositions of the present invention can be
formulated for parenteral administration, e.g., formulated for
injection via the intravenous, intramuscular, sub-cutaneous, or
even intraperitoneal routes. The preparation of an aqueous
composition that contains an OX40L antibody or inhibitor that will
be known to those of skill in the art in light of the present
disclosure. Typically, such compositions can be prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for use to prepare solutions or suspensions upon the
addition of a liquid prior to injection can also be prepared; and,
the preparations can also be emulsified.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil, or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that it may be easily injected. It also
should be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
The compositions may be formulated into a neutral or salt form.
Pharmaceutically acceptable salts, include the acid addition salts
(formed with the free amino groups of the protein) and which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing,
for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like),
suitable mixtures thereof, and vegetable oils. The prevention of
the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the
active ingredients in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques,
which yield a powder of the active ingredient, plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
An effective amount of therapeutic or prophylactic composition is
determined based on the intended goal. The term "unit dose" or
"dosage" refers to physically discrete units suitable for use in a
subject, each unit containing a predetermined quantity of the
composition calculated to produce the desired responses discussed
above in association with its administration, i.e., the appropriate
route and regimen. The quantity to be administered, both according
to number of treatments and unit dose, depends on the result and/or
protection desired. Precise amounts of the composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting dose include physical and clinical
state of the subject, route of administration, intended goal of
treatment (alleviation of symptoms versus cure), and potency,
stability, and toxicity of the particular composition. Upon
formulation, solutions will be administered in a manner compatible
with the dosage formulation and in such amount as is
therapeutically or prophylactically effective. The formulations are
easily administered in a variety of dosage forms, such as the type
of injectable solutions described above.
III. Treatment of Disease
Methods of the present invention include treatment or prevention of
inflammation and autoimmunity. Inflammation may be treated by
administering OX40L inhibitors that inhibit the differentiation of
inflammatory T cells, promote the generation and function of
regulatory T cells by inducing IL-10 and inhibiting TNF-.alpha.,
and reduce aberrant Th2 cell responses. The inflammation may be a
component of an autoimmune disease or inflammation as a result of a
non-autoimmune related dysfunction (e.g. cancer, injury, etc.). In
further instances, the inflammation may be idiopathic, or of
unknown cause.
The methods of the present invention also include the treatment or
prevention of autoimmunity by the administration of OX40L
inhibitors that eliminate or reduce aberrant T follicular helper
cell-(Tfh) responses that may contribute to the pathogenicity of
autoimmune disease. The compositions of the present invention have
been shown to have in vivo utility for the treatment of GVHD and
graft rejection.
Embodiments of the invention can be used to treat or ameliorate a
number of immune-mediated, inflammatory, or autoimmune diseases,
e.g., diabetes, graft rejection, etc. Examples of such diseases or
disorders include, but are not limited to arthritis (rheumatoid
arthritis such as acute arthritis, chronic rheumatoid arthritis,
gout or gouty arthritis, acute gouty arthritis, acute immunological
arthritis, chronic inflammatory arthritis, degenerative arthritis,
type II collagen-induced arthritis, infectious arthritis, Lyme
arthritis, proliferative arthritis, psoriatic arthritis, Still's
disease, vertebral arthritis, and juvenile-onset rheumatoid
arthritis, osteoarthritis, arthritis chronica progrediente,
arthritis deformans, polyarthritis chronica primaria, reactive
arthritis, and ankylosing spondylitis), inflammatory
hyperproliferative skin diseases, psoriasis such as plaque
psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of
the nails, atopy including atopic diseases such as hay fever and
Job's syndrome, dermatitis including contact dermatitis, chronic
contact dermatitis, exfoliative dermatitis, allergic dermatitis,
allergic contact dermatitis, dermatitis herpetiformis, nummular
dermatitis, seborrheic dermatitis, non-specific dermatitis, primary
irritant contact dermatitis, and atopic dermatitis, x-linked hyper
IgM syndrome, allergic intraocular inflammatory diseases, urticaria
such as chronic allergic urticaria and chronic idiopathic
urticaria, including chronic autoimmune urticaria, myositis,
polymyositis/dermatomyositis, juvenile dermatomyositis, toxic
epidermal necrolysis, scleroderma (including systemic scleroderma),
sclerosis such as systemic sclerosis, multiple sclerosis (MS) such
as spino-optical MS, primary progressive MS (PPMS), and relapsing
remitting MS (RRMS), progressive systemic sclerosis,
atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic
sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease
(IBD) (for example, Crohn's disease, autoimmune-mediated
gastrointestinal diseases, colitis such as ulcerative colitis,
colitis ulcerosa, microscopic colitis, collagenous colitis, colitis
polyposa, necrotizing enterocolitis, and transmural colitis, and
autoimmune inflammatory bowel disease), bowel inflammation,
pyoderma gangrenosum, erythema nodosum, primary sclerosing
cholangitis, respiratory distress syndrome, including adult or
acute respiratory distress syndrome (ARDS), meningitis,
inflammation of all or part of the uvea, iritis, choroiditis, an
autoimmune hematological disorder, rheumatoid spondylitis,
rheumatoid synovitis, hereditary angioedema, cranial nerve damage
as in meningitis, herpes gestationis, pemphigoid gestationis,
pruritis scroti, autoimmune premature ovarian failure, sudden
hearing loss due to an autoimmune condition, IgE-mediated diseases
such as anaphylaxis and allergic and atopic rhinitis, encephalitis
such as Rasmussen's encephalitis and limbic and/or brainstem
encephalitis, uveitis, such as anterior uveitis, acute anterior
uveitis, granulomatous uveitis, nongranulomatous uveitis,
phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN or idiopathic membranous nephropathy,
membrano- or membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, proliferative nephritis,
autoimmune polyglandular endocrine failure, balanitis including
balanitis circumscripta plasmacellularis, balanoposthitis, erythema
annulare centrifugum, erythema dyschromicum perstans, eythema
multiform, granuloma annulare, lichen nitidus, lichen sclerosus et
atrophicus, lichen simplex chronicus, lichen spinulosus, lichen
planus, lamellar ichthyosis, epidermolytic hyperkeratosis,
premalignant keratosis, pyoderma gangrenosum, allergic conditions
and responses, allergic reaction, eczema including allergic or
atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular
palmoplantar eczema, asthma such as asthma bronchiale, bronchial
asthma, and auto-immune asthma, conditions involving infiltration
of T cells and chronic inflammatory responses, immune reactions
against foreign antigens such as fetal A-B-O blood groups during
pregnancy, chronic pulmonary inflammatory disease, autoimmune
myocarditis, leukocyte adhesion deficiency, lupus, including lupus
nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,
extra-renal lupus, discoid lupus and discoid lupus erythematosus,
alopecia lupus, systemic lupus erythematosus (SLE) such as
cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome
(NLE), and lupus erythematosus disseminatus, juvenile onset (Type
I) diabetes mellitus, including pediatric insulin-dependent
diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type
II diabetes) and autoimmune diabetes. Also contemplated are immune
responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, sarcoidosis,
granulomatosis including lymphomatoid granulomatosis, Wegener's
granulomatosis, agranulocytosis, vasculitides, including
vasculitis, large-vessel vasculitis (including polymyalgia
rheumatica and gianT cell (Takayasu's) arteritis), medium-vessel
vasculitis (including Kawasaki's disease and polyarteritis
nodosa/periarteritis nodosa), microscopic polyarteritis,
immunovasculitis, CNS vasculitis, cutaneous vasculitis,
hypersensitivity vasculitis, necrotizing vasculitis such as
systemic necrotizing vasculitis, and ANCA-associated vasculitis,
such as Churg-Strauss vasculitis or syndrome (CSS) and
ANCA-associated small-vessel vasculitis, temporal arteritis,
aplastic anemia, autoimmune aplastic anemia, Coombs positive
anemia, Diamond Blackfan anemia, hemolytic anemia or immune
hemolytic anemia including autoimmune hemolytic anemia (AIHA),
Addison's disease, autoimmune neutropenia, pancytopenia,
leukopenia, diseases involving leukocyte diapedesis, CNS
inflammatory disorders, Alzheimer's disease, Parkinson's disease,
multiple organ injury syndrome such as those secondary to
septicemia, trauma or hemorrhage, antigen-antibody complex-mediated
diseases, anti-glomerular basement membrane disease,
anti-phospholipid antibody syndrome, allergic neuritis, Behcet's
disease/syndrome, Castleman's syndrome, Goodpasture's syndrome,
Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,
pemphigoid such as pemphigoid bullous and skin pemphigoid,
pemphigus (including pemphigus vulgaris, pemphigus foliaceus,
pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus),
autoimmune polyendocrinopathies, Reiter's disease or syndrome,
thermal injury, preeclampsia, an immune complex disorder such as
immune complex nephritis, antibody-mediated nephritis,
polyneuropathies, chronic neuropathy such as IgM polyneuropathies
or IgM-mediated neuropathy, autoimmune or immune-mediated
thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP)
including chronic or acute ITP, scleritis such as idiopathic
cerato-scleritis, episcleritis, autoimmune disease of the testis
and ovary including autoimmune orchitis and oophoritis, primary
hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as autoimmune thyroiditis, Hashimoto's
disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, polyglandular syndromes such as autoimmune
polyglandular syndromes (or polyglandular endocrinopathy
syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome
or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,
encephalomyelitis such as allergic encephalomyelitis or
encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE), experimental autoimmune encephalomyelitis,
myasthenia gravis such as thymoma-associated myasthenia gravis,
cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus
myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor
neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic
hepatitis, lupoid hepatitis, gianT cell hepatitis, chronic active
hepatitis or autoimmune chronic active hepatitis, lymphoid
interstitial pneumonitis (LIP), bronchiolitis obliterans
(non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease
(IgA nephropathy), idiopathic IgA nephropathy, linear IgA
dermatosis, acute febrile neutrophilic dermatosis, subcorneal
pustular dermatosis, transient acantholytic dermatosis, cirrhosis
such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune
enteropathy syndrome, Celiac or Coeliac disease, celiac sprue
(gluten enteropathy), refractory sprue, idiopathic sprue,
cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's
disease), coronary artery disease, autoimmune ear disease such as
autoimmune inner ear disease (AIED), autoimmune hearing loss,
polychondritis such as refractory or relapsed or relapsing
polychondritis, pulmonary alveolar proteinosis, Cogan's
syndrome/nonsyphilitic interstitial keratitis, Bell's palsy,
Sweet's disease/syndrome, rosacea autoimmune, zoster-associated
pain, amyloidosis, a non-cancerous lymphocytosis, a primary
lymphocytosis, which includes monoclonal B cell lymphocytosis
(e.g., benign monoclonal gammopathy and monoclonal gammopathy of
undetermined significance, MGUS), peripheral neuropathy,
paraneoplastic syndrome, channelopathies such as epilepsy,
migraine, arrhythmia, muscular disorders, deafness, blindness,
periodic paralysis, and channelopathies of the CNS, autism,
inflammatory myopathy, focal or segmental or focal segmental
glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,
chorioretinitis, autoimmune hepatological disorder, fibromyalgia,
multiple endocrine failure, Schmidt's syndrome, adrenalitis,
gastric atrophy, presenile dementia, demyelinating diseases such as
autoimmune demyelinating diseases and chronic inflammatory
demyelinating polyneuropathy, Dressler's syndrome, alopecia greata,
alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon,
esophageal dysmotility, sclerodactyl), and telangiectasia), male
and female autoimmune infertility, e.g., due to anti-spermatozoan
antibodies, mixed connective tissue disease, Chagas' disease,
rheumatic fever, recurrent abortion, farmer's lung, erythema
multiforme, post-cardiotomy syndrome, Cushing's syndrome,
bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, parasitic diseases
such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,
aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,
endocarditis, endomyocardial fibrosis, diffuse interstitial
pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis,
idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis,
erythema elevatum et diutinum, erythroblastosis fetalis,
eosinophilic faciitis, Shulman's syndrome, Felty's syndrome,
flariasis, cyclitis such as chronic cyclitis, heterochronic
cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis,
Henoch-Schonlein purpura, human immunodeficiency virus (HIV)
infection, SCID, acquired immune deficiency syndrome (AIDS),
echovirus infection, sepsis, endotoxemia, pancreatitis,
thyroxicosis, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, gianT cell polymyalgia, chronic hypersensitivity
pneumonitis, keratoconjunctivitis sicca, epidemic
keratoconjunctivitis, idiopathic nephritic syndrome, minimal change
nephropathy, benign familial and ischemia-reperfusion injury,
transplant organ reperfusion, retinal autoimmunity, joint
inflammation, bronchitis, chronic obstructive airway/pulmonary
disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic
disorders, asperniogenese, autoimmune hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica, enteritis allergica, erythema nodosum leprosum,
idiopathic facial paralysis, chronic fatigue syndrome, febris
rheumatica, Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary
idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis
granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma
gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,
non-malignant thymoma, vitiligo, toxic-shock syndrome, food
poisoning, conditions involving infiltration of T cells,
leukocyte-adhesion deficiency, immune responses associated with
acute and delayed hypersensitivity mediated by cytokines and
T-lymphocytes, diseases involving leukocyte diapedesis, multiple
organ injury syndrome, antigen-antibody complex-mediated diseases,
antiglomerular basement membrane disease, allergic neuritis,
autoimmune polyendocrinopathies, oophoritis, primary myxedema,
autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic
diseases, nephrotic syndrome, insulitis, polyendocrine failure,
autoimmune polyglandular syndrome type I, adult-onset idiopathic
hypoparathyroidism (AOIH), cardiomyopathy such as dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA),
hemochromatosis, myocarditis, nephrotic syndrome, primary
sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or
chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid
sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,
Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary eosinophilia, bronchopneumonic aspergillosis,
aspergilloma, or granulomas containing eosinophils, anaphylaxis,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome,
angiectasis, autoimmune disorders associated with collagen disease,
rheumatism, neurological disease, lymphadenitis, reduction in blood
pressure response, vascular dysfunction, tissue injury,
cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral
ischemia, and disease accompanying vascularization, allergic
hypersensitivity disorders, glomerulonephritides, reperfusion
injury, ischemic re-perfusion disorder, reperfusion injury of
myocardial or other tissues, lymphomatous tracheobronchitis,
inflammatory dermatoses, dermatoses with acute inflammatory
components, multiple organ failure, bullous diseases, renal
cortical necrosis, acute purulent meningitis or other central
nervous system inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated
syndromes, cytokine-induced toxicity, narcolepsy, acute serious
inflammation, chronic intractable inflammation, pyelitis,
endarterial hyperplasia, peptic ulcer, valvulitis, graft versus
host disease, contact hypersensitivity, asthmatic airway
hyperreaction, myasthenia gravis, immune thrombocytopenic purpura,
antineutrophil cytoplasmic autoantibody-mediated disease,
IgA-mediated vasculitis, Ig4-related disorders, and
endometriosis.
IV. In Vitro or Ex Vivo Administration
As used herein, the term in vitro administration refers to
manipulations performed on cells removed from or outside of a
subject, including, but not limited to cells in culture. The term
ex vivo administration refers to cells which have been manipulated
in vitro, and are subsequently administered to a subject. The term
in vivo administration includes all manipulations performed within
a subject, including administrations.
In certain aspects of the present invention, the compositions may
be administered either in vitro, ex vivo, or in vivo. In certain in
vitro embodiments, autologous T cells are incubated with
compositions of this invention. The cells can then be used for in
vitro analysis, or alternatively for ex vivo administration.
V. Combination Therapy
The compositions and related methods of the present invention,
particularly administration of an OX40L antibody or antigen binding
fragment may also be used in combination with the administration of
traditional therapies. These include, but are not limited to, the
administration of immunosuppressive or immunomodulating therapies
or treatments.
In one aspect, it is contemplated that an OX40L antibody or antigen
binding fragment is used in conjunction with an additional therapy.
Alternatively, antibody administration may precede or follow the
other treatment by intervals ranging from minutes to weeks. In
embodiments where the other agents are administered separately, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent and
antibody would still be able to exert an advantageously combined
effect on the subject. In such instances, it is contemplated that
one may administer both modalities within about 12-24 h of each
other and, more preferably, within about 6-12 h of each other. In
some situations, it may be desirable to extend the time period for
administration significantly, however, where several days (2, 3, 4,
5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations.
Administration of the OX40L antibody or antigen binding fragment of
the present invention to a patient/subject will follow general
protocols for the administration of such compounds, taking into
account the toxicity, if any. It is expected that the treatment
cycles would be repeated as necessary. It also is contemplated that
various standard therapies, such as hydration, may be applied in
combination with the described therapy.
TABLE-US-00001 VI. Sequence Listing SEQ ID NO: 1-mouse
anti-hOX40L_19A3 (9295)- antibody heavy chain mIgGH2b
ATGGAATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGT
ACACTCTGAGGTCCAGCTTCAGCAGTCTGGGCCTGAGCTGGGGCAGCCTG
GGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGT
TACAGCATGCACTGGGTGAAGCAGAGCCATAGGAAAAGCCCTGAGTGGAT
TGGAAAAATTGATCCTTACAATGGTGTGACTACCTATAATCAGAGGTTCA
CGGGCAAGGCCACATTGACTGTCGACACATCTTCCAGCACAGCCTACATG
CATCTCAACAGCCTGACATCTGAGGACTCTGCAATCTTTTACTGTGCGAG
AGAGGGGTTTGCTTATTGGGGCCAAGGGACTCTGGTCTCTGTCTCTGAAG
CCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGAT
ACAACTGGTTCCTCCGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCC
TGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGC
ACACMTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCTCA
GTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGT
TGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCG
GGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAA
TGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCC
AAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTG
TGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTT
GTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGA
TTACAACAGTACTATCCGGGTGGTCAGCACCCTCCCCATCCAGCACCAGG
ACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTC
CCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGC
TCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAG
ATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGT
GTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGC
ACCAGTCCTGGACTCTGACGGTTCTTACTTCATATATAGCAAGCTCAATA
TGAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGA
CACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCC
GGGTAAAGCTAGCTGAAAAA (SEQ ID NO: 1) SEQ ID NO: 2-mouse
anti-hOX40L_19A3 (9295)- antibody heavy chain mIgGH2b. The
classical TTXP signature is underlined
MEWSWIFLFLLSGTAGVHSEVQLQQSGPELGQPGASVKISCKASGYSFTG
YSMHWVKQSHRKSPEWIGKIDPYNGVITYNQRFTGKATLTVDTSSSTAYM
HLNSLTSEDSAIFYCAREGFAYWGQGTLVSVSEAKTTPPSVYPLAPGCGD
TTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSS
VTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHK
CPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWF
VNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDL
PSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLICLVVGFNPGDIS
VEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVR
HEGLKNYYLKKTISRSPGKAS (SEQ ID NO: 2) SEQ ID NO: 3-the leader
peptide of the VH region of mouse anti-hOX40L_19A3 (9295)-antibody
heavy chain mIgGH2b: MEWSWIFLFLLSGTAGVHS (SEQ ID NO: 3) SEQ ID NO:
4 Variable region of mouse anti- hOX40L_19A3 (9295)-antibody heavy
chain mIgGH2b EVQLQQSGPELGQPGASVKISCKASGYSFTGYSMHWVKQSHRKSPEWIGK
IDPYNGVTTYNQRFTGKATLTVDTSSSTAYMHLNSLTSEDSAIFYCAREG FAYWGQGTLVSVSEAK
SEQ ID NO: 4) SEQ ID NO: 5-CDR1 of the VH region of mouse anti-
hOX40L_19A3 (9295)-antibody heavy chain mIgGH2b GYSFTGYSMH (SEQ ID
NO: 5) SEQ ID NO: 6-CDR2 of the VH region of mouse anti-
hOX40L_19A3 (9295)-antibody heavy chain mIgGH2b KIDPYNGVTTYNQRFTG
(SEQ ID NO: 6) SEQ ID NO: 7-CDR3 of the VH region of mouse anti-
hOX40L_19A3 (9295)-antibody heavy chain mIgGH2b EGFAY (SEQ ID NO:
7) SEQ ID NO: 8-mouse anti-hOX40L_19A3 (9295)- antibody kappa light
chain mIgK ATGCATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGT
CATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGT
CTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCTCCTCAAGT
GTCCGTTATATTCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACT
CTTGATTTATAGCACATCCGACCTGGCTTCTGGAGTCCCTGCTCGCTTCA
GTGGCGGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAG
GCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGACTGGTTACCCGCT
CACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCAC
CAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGT
GCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGT
CAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTT
GGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTC
ACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGC
CACTCACAAGGCATCAACTTCACCCATCGTCAAGAGCTTCAACAGGAATG AGTGTTAGAAAA
(SEQ ID NO: 8) SEQ ID NO: 9-mouse anti-hOX40L_19A3 (9295)- antibody
kappa light chain mIgK. The LE classical signature is underlined
MHFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASPGEKVTITCSASSS
VRYIHWFQQKPGTSPKLLIYSTSDLASGVPARFSGGGSGTSYSLTISRME
AEDAATYYCQQRTGYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGG
ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTL
TLIKDEYERHNSYTCEATHKASTSPIVKSFNRNEC (SEQ ID NO: 9) SEQ ID NO:
10-the leader peptide of the VL region of mouse anti-hOX40L_19A3
(9295)-antibody kappa light chain mIgK: MHFQVQIFSFLLISASVIMSRG (SEQ
ID NO: 10) SEQ ID NO: 11 Variable region of mouse anti- hOX40L_19A3
(9295)-antibody kappa light chain mIgK
QIVLTQSPAIMSASPGEKVTITCSASSSVRYIHWFQQKPGTSPKLLIYST
SDLASGVPARFSGGGSGTSYSLTISRMEAEDAATYYCQQRTGYPLTFGAG TK (SEQ ID NO:
11) SEQ ID NO: 12-CDR1 of the VL region of mouse anti- hOX40L_19A3
(9295)-antibody kappa light chain mIgK SASSSVRYIH (SEQ ID NO: 12)
SEQ ID NO: 13-CDR2 of the VL region of mouse anti- hOX40L_19A3
(9295)-antibody kappa light chain mIgK STSDLAS (SEQ ID NO: 13) SEQ
ID NO: 14-CDR3 of the VL region of mouse anti-hOX40L_19A3
(9295)-antibody kappa light chain mIgK QQRTGYPLT (SEQ ID NO: 14)
SEQ ID NO: 15-DNA sequence of Clone 5C6 (8703) Hybridoma OX40L
antibody heavy chain; chimeric on hIgG4
ATGAAGTGCTCCTGGGTCATCTTCTTCCTCATGGCCGTGGTGACCGGAGT
GAACTCTGAGGTGCAACTCCAGCAGTCAGGAGCTGAAATCGTGAAGCCAG
GCGCAAGTGTGAAGCTGTCCTGCACCGCTTCTGGGTTCAACATCAAGGAC
ACCTACATGCACTGGGTGAAGCAGCGGCCAGAGCAGGGGTTGGAGTGGAT
TGGCAGAATTGACCCTAGGAACGACAACACCAAGTTTGACCCTAAGTTTC
GCGGGAAAGCAACACTGACTGCCGATACATCCAGCAATACTGCCTACCTG
CAGCTGAGCAGCCTTACATCCGAGGATGCCGCCGTCTACTACTGTGTGCC
CGTCCCCACAAGGAGCTGGTATTTTGATGTGTGGGGGGCCGGCACTAGCG
TCACAGTCTCCAGCGCCAAAACAAAGGGCCCATCCGTCTTCCCCCTGGCG
CCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC
TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAA
GACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACA
AGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCT
GAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA
CACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCA
GGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAG CTGA (SEQ ID NO:
15) SEQ ID NO: 16-Amino Acid sequence of Clone 5C6 (8703) Hybridoma
OX40L antibody heavy chain; the classical TXXP signature is
underlined MKCSWVIFFLMAVVTGVNSEVQLQQSGAEIVKPGASVKLSCTASGFNIKD
TYMHWVKQRPEQGLEWIGRIDPRNDNTKFDPKFRGKATLTADTSSNTAYL
QLSSLTSEDAAVYYCVPVPTRSWYFDVWGAGTSVTVSSAKTKGPSVFPLA
PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI
EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL
HNHYTQKSLSLSLGKAS (SEQ ID NO: 16) SEQ ID NO: 17-Signal sequence of
Clone 5C6 (8703) Hybridoma OX40L antibody heavy chain
MKCSWVIFFLMAVVTGVNS (SEQ ID NO: 17) SEQ ID NO: 18-Variable region
of Clone 5C6 (8703) Hybridoma OX40L antibody heavy chain
EVQLQQSGAEIVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGR
IDPRNDNTKFDPKFRGKATLTADTSSNTAYLQLSSLTSEDAAVYYCVPVP
TRSWYFDVWGAGTSVTVSSAK (SEQ ID NO: 18) SEQ ID NO: 19-CDR1 of Clone
5C6 (8703) Hybridoma OX40L antibody heavy chain GFNIKDTYMH (SEQ ID
NO: 19) SEQ ID NO: 20-CDR2 of Clone 5C6 (8703) Hybridoma OX40L
antibody heavy chain RIDPRNDNTKFDPKFRG (SEQ ID NO: 20) SEQ ID NO:
21-CDR3 of Clone 5C6 (8703) Hybridoma OX40L antibody heavy chain
VPTRSWYFDV (SEQ ID NO: 21) SEQ ID NO: 22-DNA sequence of Clone 5C6
(8703) Hybridoma OX40L antibody light chain; chimeric on hIgGK-C
backbone. ATGGAGACCCATTCCCAAGTGTTCGTCTACATGCTGCTCTGGCTCTCCGG
AGTCGAAGGAGACATCGTGATGACCCAGTCTCACAAGTTCATGTCCACCA
GCGTGGGCGATAGAGTGTCTATTACCTGCAAGGCCTCACAGGACGTGGGG
AAATCCGTCGTGTGGTTTCAGCAGAAGCCTGGCCAGAGTCCAAAGCTTTT
GATCTACTGGGCAAGCACCAGGCACACAGGGGTGCCCGATCGGTTTACAG
GCAGCGGGAGCGGCACTGATTTTACTCTGACAATTTCCAACGTCCAGAGC
GAGGACCTGGCTAATTATTTCTGTCAGCAGTACACTAGCTACCCCTACAT
GTACACATTCGGGGGGGGCACAAAGCTCGAGATCAAACGAACTGTGGCTG
CACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA
AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA
GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC
CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGA
AGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG
GAGAGTGTGCTAGCTGA (SEQ ID NO: 22) SEQ ID NO: 23-Amino Acid sequence
of Clone 5C6 (8703) Hybridoma OX40L antibody light chain; the LE
classical signature is underlined. chimeric on hIgGK-C backbone
METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSVGDRVSITCKASQDVG
KSVVWFQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQS
EDLANYFCQQYTSYPYMYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST
LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS (SEQ ID NO: 23) SEQ ID NO:
24-Signal sequence of Clone 5C6 (8703) Hybridoma OX40L antibody
light chain METHSQVFVYMLLWLSGVEG (SEQ ID NO: 24) SEQ ID NO:
25-Variable region of Clone 5C6 (8703) Hybridoma OX40L antibody
light chain DIVMTQSHKFMSTSVGDRVSITCKASQDVGKSVVWFQQKPGQSPKLLIYW
ASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLANYFCQQYTSYPYMYTF GGGTK (SEQ ID
NO: 25) SEQ ID NO: 26-CDR1 of Clone 5C6 (8703) Hybridoma OX40L
antibody light chain KASQDVGKSVV (SEQ ID NO: 26) SEQ ID NO: 27-CDR2
of Clone 5C6 (8703) Hybridoma OX40L antibody light chain WASTRHT
(SEQ ID NO: 27) SEQ ID NO: 28-CDR3 of Clone 5C6 (8703) Hybridoma
OX40L antibody light chain QQYTSYPYMYT (SEQ ID NO: 28) SEQ ID NO:
29-DNA sequence of Clone 44F3 (8704) Hybridoma OX40L antibody heavy
chain chimeric on hIgG4
ATGGAGAGACACTGGATCTTGCTCCTGCTGTTGTCCGTGACCGCTGGAGT
CCATAGCCAGGTCCAACTGCAACAGTCCGGAGCAGAACTTGCTAGGCCTG
GAGCAAGCGTCAAAATGTCCTGTAAGGCTTCCGGATACACCCTCGCAAGC
TACACCCTGCACTGGGTGAAGCAGCGCCCTGGGCAGGGGCTTGAATGGAT
TGGCTATATTAATCCCAACAGTGGCTATACCAACTACATCCAGAAGTTCA
AGGACAAGGCCACCCTCACAGCCGACAAGAGCTCATCAACTGCTTACATG
CAGCTGAGTTCTCTGACATCTGAGGACAGTGCCGTGTACTACTGCGCTAA
AGGCGGCGGGGATCGGTATTGTACAGATTGCGCCATGGATTATTGGGGCC
AGGGCACATCTGTGACTGTGTCTCCCGCCAAAACAAAGGGCCCATCCGTC
TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCT
GGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGA
ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG
TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAG
CTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA
CCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCC
TGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCC
AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG
TGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC
GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG
ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTC
CCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
GCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACC
AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC
TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCG
TGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCT GGGTAAAGCTAGCTGA
(SEQ ID NO: 29) SEQ ID NO: 30-Amino Acid sequence of Clone 44F3
(8704) Hybridoma OX40L antibody heavy chain chimeric on hIgG4; the
classical TXXP signature is underlined
MERHWILLLLLSVTAGVHSQVQLQQSGAELARPGASVKMSCKASGYTLAS
YTLHWVKQRPGQGLEWIGYINPNSGYTNYIQKFKDKATLTADKSSSTAYM
QLSSLTSEDSAVYYCAKGGGDRYCTDCAMDYWGQGTSVTVSPAKTKGPSV
FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM
HEALHNHYTQKSLSLSLGKAS (SEQ ID NO: 30) SEQ ID NO: 31-Signal sequence
of Clone 44F3 (8704) Hybridoma OX40L antibody heavy chain
MERHWILLLLLSVTAGVHS (SEQ ID NO: 31) SEQ ID NO: 32-Variable region
of Clone 44F3 (8704) Hybridoma OX40L antibody heavy chain
QVQLQQSGAELARPGASVKMSCKASGYTLASYTLHWVKQRPGQGLEWIGY
INPNSGYTNYIQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCAKGG
GDRYCTDCAMDYWGQGTSVTVSPAK (SEQ ID NO: 32) SEQ ID NO: 33-CDR1 of
Clone 44F3 (8704) Hybridoma OX40L antibody heavy chain GYTLASYTLH
(SEQ ID NO: 33) SEQ ID NO: 34-CDR2 of Clone 44F3 (8704) Hybridoma
OX40L antibody heavy chain YINPNSGYTNYIQKFKD (SEQ ID NO: 34) SEQ ID
NO: 35-CDR3 of Clone 44F3 (8704) Hybridoma OX40L antibody heavy
chain GGGDRYCTDCAMDY (SEQ ID NO: 35) SEQ ID NO: 36-DNA sequence of
Clone 44F3 (8704) Hybridoma OX40L antibody light chain; chimeric on
hIgGK-C backbone ATGCACTCCCTTGCACTTCTGTTGAGCCTCTTGCTGCTGTGCGTGAGTGA
CAGCAGAGCTGAGACCACCGTGACACAGTCTCCTGCCTCTCTGTCAATGA
CCATCGGAGAAAAGGTGACCATCAGGTGCATGACTAGCATCGACATTGAC
GATGATATGAACTGGTACCAGCAGAAGCCAGGGGAGCCTCCAAAGCTGCT
GATTTCCGAGGGAAAGACACTCCGCCCCGGGGTCCCCAGTCGGTTTTCC
AGCTCCGGGTACGGCACTGACTTTGTCTTCACTATTGAGAACATGCTCAG
CGAGGATGTGGCCGATTACTATTGTCTCCAAAGCGACAATCTGCCCTTCA
CATTCGGCTCCGGCACAAAACTCGAGATCAAACGAACTGTGGCTGCACCA
TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC
CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC
ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCA
CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTGCTAGCTGA
(SEQ ID NO: 36) SEQ ID NO: 37-Amino Acid sequence of Clone 44F3
(8704) Hybridoma OX40L antibody light chain chimeric onhIgGK-C; the
LE classical signature is underlined
MHSLALLLSLLLLCVSDSRAETTVTQSPASLSMTIGEKVTIRCMTSIDID
DDMNWYQQKPGEPPKLLISEGKTLRPGVPSRFSSSGYGTDFVFTIENMLS
EDVADYYCLQSDNLPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA
SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS (SEQ ID NO: 37) SEQ ID NO:
38-Signal sequence of Clone 44F3 (8704) Hybridoma OX40L antibody
light chain MHSLALLLSLLLLCVSDSRA (SEQ ID NO: 38) SEQ ID NO:
39-Variable region of Clone 44F3 (8704) Hybridoma OX40L antibody
light chain ETTVTQSPASLSMTIGEKVTIRCMTSIDIDDDMNWYQQKPGEPPKLLISE
GKTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPFTFGS GTK (SEQ ID NO:
39) SEQ ID NO: 40-CDR1 of Clone 44F3 (8704) Hybridoma OX40L
antibody light chain MTSIDIDDDMN (SEQ ID NO: 40) SEQ ID NO: 41-CDR2
of Clone 44F3 (8704) Hybridoma OX40L antibody light chain EGKTLRP
(SEQ ID NO: 41) SEQ ID NO: 42-CDR3 of Clone 44F3 (8704) Hybridoma
OX40L antibody light chain LQSDNLPFT (SEQ ID NO: 42)
VII. Examples
The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1: Characterization of OX40L Antibodies
The OX40L antibodies, 5C6 (also labeled as AB104_105.5C6.3F9), 19A3
(also labeled as AB104_105.19A3.2C4), and 44F3 (also labeled as
AB104_105.44F3.2F7) were tested for their ability to promote
cytokine production by naive T cells co-cultured with mDCs using
the following assays. The effect of the OX40L antibodies on the
proliferation and viability of mDC cultures were also tested using
the assays described in this example.
The OX40L antibody clones 5C6, 19A3, and 44F3 were found to 1)
recognize unique epitopes on human OX40L, 2) inhibit the
differentiation of IL-10 low/TNFa high producing inflammatory Th2
primed by TSLP-mDCs, 3) inhibit the proliferation and the
production of TNF-a, and promote IL-10 by CD4 T cells cultured with
OX40L-transfected cell line. These results are shown in FIGS.
1-8.
TSLP-mDCs and Naive CD4 T Cell Cocluture--Isolation and Culture of
Blood Myeloid DCs (mDCs).
PBMCs were isolated form buffy coats or apheresis blood samples
obtained from adult healthy volunteers. mDCs (lineage.sup.-,
CD4.sup.+, CD11c.sup.+) were enriched from the PBMCs by using Human
pan-DC pre-enrichment kit (STEMCELL#19251) or Human myeloid DC
enrichment kit (STEMCELL#19061) according to methods known in the
art. The enriched mDCs were stained with FITC-CD3 (#349201), CD14
(#347493). CD16 (#555406), CD19 (#555412), CD20 (#555622), CD56
(#3032769), APC-CD11c (#340544), and V450-CD4 (#2342693) (all
antibodies are from BD), and then linage.sup.- CD11c.sup.+CD4.sup.+
population was sorted as myeloid DCs by a FACS Aria2 (BD
Biosciences). mDCs were cultured in RPMI+GlutaMAX (gibco), 10 mM
HEPES (gibco), Penicillin/Streptmycin/L-Glutamine (gibco), 1 mM
Sodium Pyruvate (SIGMA) and MEM Non-Essential Amino Acids Solution
(Hyclone) containing 10% human AB serum (GemCell#100-512). Cells
were seeded at a density of 1.about.2.times.10.sup.5/200 ul medium
in flat-bottomed 96-well plate in the presence of 20 ng/ml
recombinant human TSLP. The recombinant human TSLP had been
prepared in-house using an adenovirus vector system as described
previously (Soumelis, et al., 2002). After incubation for
20.about.24 hours, mDCs are harvested and washed with culture
medium.
Isolation of Naive T Cells.
PBMCs were isolated form buffy coats blood or apheresis blood
samples obtained from adult healthy volunteers. CD4.sup.+ naive T
cells were enriched from the PBMCs using Human CD4.sup.+ T cell
Enrichment Cocktail (STEMCELL#15062) and Human Naive CD4.sup.+ T
cell Enrichment kit (STEMCELL#19155). The enriched CD4.sup.+ naive
T cells were stained with FITC-CD8 (#340692), CD14 (#347493), CD16
(#555406), CD19 (#555412), CD20 (#555622), CD25 (#340694), CD56
(#3032769), BDCA-2 (130-090-510; Miltenyi Biotech), CD11c
(#3011791), TCR.gamma..delta. (#347903), PE-CCR7 (#353204;
BioLegend), PE-Cy7-CD45RO (#337168), APC-CD45RA (#304112;
BioLegend), and V450-CD4 (#2342693) (all antibodies are from BD
except BDCA-2, CCR7 and CD45RA). The
linage.sup.-CD4.sup.+CD45RA.sup.+CD45RO.sup.-CCR7.sup.+ population
was sorted as naive CD4.sup.+ T cells by a FACS Aria2.
DC and T Cell Coculture.
Freshly purified allogeneic naive CD4.sup.+ T cells
(2.5.times.10.sup.4 cells per well) were cocultured with TSLP DCs
(5.times.10.sup.3 cells per well) at DC/T cell ratio 1:5 in
round-bottom 96-well culture plates in the presence of 50 ug/ml
anti-OX40L mAb (ik-5, provided by Dr. Toshiyuki Hori), in-house
anti-OX40L mAbs (5C6, 19A3, and 44F3). Mouse IgG2a (16-4724-85;
eBioscience) and IgG2b (MAB004; R&D systems) were used as
controls. After 6-8 days culture, naive T cells were harvested and
re-stimulated with immobilized anti-CD3 (OKT3: 1 ug/ml) plus
anti-CD28 (1 ug/ml) at a concentration of 10.sup.5 cells/100 ul in
flat-bottom 96-well plates for 20.about.24 hours. The production of
IL-4, IL-5, IL-10, IL-13, TNF-.alpha. and IFN-.gamma. were measured
by ELISA (all kits from R&D Systems) or Luminex
(Milliopore).
Isolation of Bulk CD4.sup.+ T Cells.
Bulk CD4.sup.+ T cells were isolated from the buffy coat of blood
from healthy adult volunteers by using Human CD4.sup.+ T cell
Enrichment Cocktail (STEMCELL#15062) followed by cell sorting as a
CD4 Lineage fraction using FITC-CD8 (#340692), CD14 (#347493), CD16
(#555406), CD19 (#555412), CD20 (#555622), CD25 (#340694), CD56
(#3032769), BDCA-2 (130-090-510; Miltenyi Biotech), CD11c
(#3011791), TCR.gamma..delta. (#347903), and APC-Cy7 CD4 (#) (all
antibodies are from BD except BDCA-2). After sorting, CD4.sup.+ T
cell were labeled by CSFE as the manufacture instructions
L Cells and T Cell Coculture.
Before the coculture with T cells, 0.2 ug/ml of anti-CD3 antibody
(OKT3) were put in the pre-seeded L cells and incubated for
1.about.2 hours. Then the CSFE-labeled CD4.sup.+ T cells were added
and cultured on the L cells in the presence of 1 ug/ml of anti-CD28
antibody (CD28.2, BD pharmingen) for 6 days. Activated CD4.sup.+ T
cell were re-stimulated with immobilized anti-CD3 (OKT3: 1 ug/ml)
plus anti-CD28 (1 ug/ml) at a concentration of 10.sup.5 cells/100
ul in flat-bottom 96-well plates for 24 hours. The production of
IL-4, IL-5, IL-10, IL-13, TNF-.alpha. and IFN-.gamma. were measured
by ELISA (all kits from R&D Systems) or Luminex
(Milliopore).
Example 2: OX40 Ligand Contributes to Human Lupus Pathogenesis by
Promoting T Follicular Helper Response
Systemic lupus erythematosus (SLE) is a chronic systemic
inflammatory autoimmune disease characterized by a breakdown of
tolerance to nuclear antigens (Tsokos, 2011). SLE displays
considerable heterogeneity in clinical manifestations and disease
course. A more comprehensive understanding of SLE pathogenesis is
long overdue; in the past 50 years, only one new drug has been
approved for SLE treatment (Murphy et al., 2013; Stohl et al.,
2012). Genome-wide association studies (GWAS) have identified many
susceptibility loci for SLE, confirming that SLE patients display
predisposing genetic factors (Cunninghame Graham et al., 2008;
Delgado-Vega et al., 2009; Gateva et al., 2009; Han et al., 2009;
International Consortium for Systemic Lupus Erythematosus et al.,
2008). The interactions of the immune system with predisposing
factors with environmental factors cause alterations in the
functions of antigen presenting cells (APCs) and lymphocytes in
SLE. APCs including dendritic cells (DCs) are aberrantly activated
in SLE patients, and promote the activation of autoreactive T and B
cells (Blanco et al., 2001; Blanco et al., 2008). The developed
autoreactive plasma cells produce pathogenic autoantibodies
directed against nuclear components and cause tissue injury.
Numerous studies with murine models have demonstrated that T
follicular helper cells (Tfh) (Craft, 2012; Linterman et al., 2009;
Vinuesa et al., 2005), a CD4.sup.+ helper T (Th) cell subset
specialized for provision of help to B cells (Crotty, 2011), play a
major pathogenic role in lupus. For example, sanroque mice that
display single recessive defect in Roquin gene develop a lupus-like
autoimmune disease by generating excessive Tfh cell responses
(Linterman et al., 2009; Vinuesa et al., 2005). Tfh cells are
essential for the formation of germinal centers (GCs), the site for
the selection of high-affinity B cells and for the development of B
cell memory (MacLennan, 1994; Vinuesa and Cyster, 2011). Tfh cells
are equipped with multiple features required for B cell help
(Crotty, 2011; King et al., 2008). IL-21 secreted by Tfh cells and
their precursors (Bentebibel et al., 2011; Bryant et al., 2007;
Chtanova et al., 2004) potently promotes the growth,
differentiation, and class-switching of B cells (Spolski and
Leonard, 2008). Inducible co-stimulator (ICOS) is highly expressed
by GC Tfh cells and mediates the interaction with B cells (Crotty,
2011; King et al., 2008; Xu et al., 2013). CD40 ligand (CD40L)
expressed by Tfh cells provides signals to B cells through CD40 for
their differentiation and class-switching (Banchereau et al.,
1994). The importance of these Tfh molecules in lupus pathogenesis
is underscored by the observations in lupus mouse models that
inhibition of the function of CD40L (Boumpas et al., 2003; Kalled
et al., 1998), ICOS (Odegard et al., 2008), IL-21 and/or IL-21
receptor (Bubier et al., 2009; Herber et al., 2007) delays the
disease course and improves the clinical symptoms. Furthermore, an
inhibition of the generation of Tfh cells in lupus prone sanroque
mice model by deleting SAP molecule abrogates the development of
renal pathology by inhibiting the (Linterman et al., 2009). These
studies provide a strong rationale to suppress the generation
and/or activity of Tfh cells for the prevention of lupus disease
from subjects with susceptible loci and/or for the treatment of
lupus patients.
In human lupus, a majority of IgG class autoantibody-producing B
cells are somatically mutated (Tiller et al., 2007), suggesting
that they are derived from GCs through interactions with Tfh cells.
Multiple studies show that the frequency of blood Tfh cells with
active phenotype is increased in active SLE patients (He et al.,
2013; Simpson et al., 2000). Furthermore, Tfh cells are also found
in T-cell and B-cell aggregates and ectopic germinal centers in the
kidneys of patients with lupus nephritis (Chang et al., 2011;
Liarski et al., 2014). These observations support the pathogenic
role of Tfh cells in human lupus. However, the mechanisms involved
in increased Tfh response in SLE patients remains unknown.
Here it is shown that OX40 ligand (OX40L) expressed by myeloid APCs
contributes to the aberrant Tfh response in SLE. OX40L was
expressed by myeloid APCs in blood as well as in inflamed tissues
in adult and pediatric active SLE patients. OX40L stimulation
induced human CD4.sup.+ T cells to express Tfh-associated
molecules, and was sufficient to induce them to become functional B
cell helpers. Finally, it is shown here that immune complexes (ICs)
containing ribonucleoprotein (RNP) present in lupus sera induce
OX40L expression by myeloid APCs through activation of TLR7. Thus,
this study shows that the RNP IC-OX40L axis likely provides an
amplification loop of the generation of autoantibodies in SLE.
Materials and Methods
Patient Samples--
Adult SLE patients (total 61:53 female and 8 male) and pediatric
SLE patients (total 38: 34 female and 4 male) who met the American
College of Rheumatology revised criteria for SLE (Hochberg, 1997)
were enrolled. All clinical and biologically relevant information
of the patients is shown in Tables 1-2 below. Clinical disease
activity was assessed using the SLE Disease Activity Index
(SLEDAI). Active patients were defined as SLEDAI score .gtoreq.6.
For adult SLE samples, blood samples from routine lab analysis were
used after informed consent was obtained. For pediatric SLE
samples, the study was approved by the Institutional Review Board
of Baylor Research Institute and informed consent was obtained from
all the participants or their parents. Control PBMCs were obtained
either from buffy coat, blood draw, or apheresis blood samples from
adult volunteers.
TABLE-US-00002 TABLE 1 Clinical and laboratory paramers of adult
SLE patients included in the study. Patient Corticotherapy
Associated number Age Sex Clinical failure Flare (dose, mg/day)
treatment SLEDAI 1 54 M A, C, H , R HCQ 2 2 27 F A, C, H, N, PP,
APS A 10 MTX, RTX 8 3 35 F A, H A 20 HCQ 4 4 33 F C, H, APS 10 HCQ,
MMF 0 4 34 F C, H, APS 10 HCQ, MMF 2 5 35 F A, C, R 7 HCQ, AZA 2 6
35 F A, C, H, R, APS 50 CYC 4 7 41 F A, C, H, R N, R 10 MPA 26 8 34
M A, C, H, R C, R, V 20 9 40 F A, C, H HCQ, MTX 0 10 32 F A, C, H,
R A, C, R HCQ 22 10 31 F A, C, H, R 25 HCQ, CYC 4 10 31 F A, C, H,
R R 25 HCQ, CYC 8 10 31 F A, C, H, R R 20 HCQ, CYC 8 11 41 F A, H,
APS 3 HCQ, MMF 4 12 72 M C, H, N, PP, R C, N, PP, R HCQ 24 13 36 F
A, C, R 10 AZA 4 14 22 F A, C, R AZA 2 15 22 F A, C, H, R R 2.5 MMF
12 16 23 M H, R R MMF 8 16 22 M H, R MMF 4 17 60 M A, H, N, R A, N,
R 10 HCQ 19 17 58 M A, H, N, R 10 HCQ 2 17 59 M A, H, N, R 5 HCQ 2
18 25 F A, C, H, PP, APS 30 HCQ, RTX 4 18 25 F A, C, H, PP, APS 25
HCQ, RTX 4 18 25 F A, C, H, PP, APS A 40 HCQ, RTX 8 19 18 F A, C,
H, R A, C, N, R 20 HCQ, MMF 24 19 19 F A, C, H, R 30 MPA 0 19 19 F
A, C, H, R 7 MPA 0 19 20 F A, C, H, R A 6 MPA 8 19 20 F A, C, H, R
6 HCQ, MPA 2 20 40 F A, C, R, APS 6 MMF 2 21 58 F A, C, H 3 HCQ,
MTX 4 22 42 F A, H, R R, V 33 23 44 M A, H, PP, R 8 MMF 4 24 18 F H
3 25 27 F A, C, R A 10 HCQ, MMF 8 26 48 F D, H, M, PP, R R 40 AZA
16 26 46 F D, H, M, PP, R 20 MMF 4 26 46 F D, H, M, PP, R 5 HCQ,
MMF 4 27 39 F A, H, N, R, APS 5 HCQ, AZA 2 28 35 F A, C, H, N A 10
8 28 35 F A, C, H, N A 15 8 28 34 F A, C, H, N 7.5 AZA 4 29 58 F A,
H A 10 AZA 8 29 57 F A, H A 15 HCQ, AZA 6 30 35 F A, C, H, M, N, R
A, R 20 HCQ, MMF 16 30 35 F A, C, H, M, N, R 17.5 HCQ, MMF 2 31 58
F A, C 17.5 HCQ 2 32 25 F A, H MTX 4 32 25 F A, H A 10 MTX 8 33 38
F A, H, R R 16 33 39 F A, H, R 8 MMF 2 34 17 M A, C, PP, R R 10
HCQ, MMF 12 35 21 F A, C, APS HCQ 4 35 21 F A, C, APS HCQ 4 35 21 F
A, C, APS HCQ 4 36 19 F A, C, R R 5 HCQ, MMF 8 37 39 F A, C, R 3
MMF, RTX 2 38 51 F A, H, R A 10 Abatacept 8 38 51 F A, H, R R 8
Abatacept 8 38 52 F A, H, R R 9 Abatacept 12 38 52 F A, H, R A 10 8
39 57 F A, H, PP, R R 5 HCQ 17 39 56 F A, H, PP, R R 20 HCQ 4 40 65
F H, APS 0 41 38 F A, C, PP C 5 HCQ 6 42 34 F A, C, H, N 10 HCQ,
MMF 4 43 28 F R, H, A R 5 HCQ, MMF 9 44 63 F C, A, R H, A 30 HCQ,
AZA 22 45 22 F C, A PP 30 HCQ, CYC 23 46 37 M A, R, D 5 MMF 2 47 37
F A 7 HCQ 2 48 57 F A 5 HCQ 4 49 53 F A 5 MTX 3 50 80 F A, N, PP 9
4 51 20 F R HCQ, MTX 2 52 41 F C, A, R 8 53 43 F A A 20 8 54 50 F H
HCQ 4 55 55 F A, C MMF 4 56 27 F A, R 16 57 25 F A, R, H, PP 23 58
18 F A, H A 12.5 AZA 8 59 35 F A 10 HCQ 4 60 37 F A, C A 10 HCQ 14
61 41 F A, C, H A, R 5 HCQ 25
TABLE-US-00003 TABLE 2 Clinical and laboratory paramers of
pediatric SLE patients included in the study. Cortico- therapy
Patient (dose, Associated number Age Sex Flare mg/day) treatment
SLEDAI 1 18 F HCQ, MPA 0 2 17 F 9 HCQ, MPA 2 3 17 F R HCQ, MPA 10 4
17 F 10 HCQ, MMF 5 5 15 F MPA 2 6 12 F R 10 HCQ, MMF 14 7 16 F HCQ,
MMF 2 8 15 F HCQ, MPA 4 9 14 F HCQ, MPA 4 10 17 F R HCQ, MPA 6 11
16 F 5 HCQ, MPA 4 12 15 M 10 HCQ, MMF 0 13 17 F 5 HCQ, MPA 0 14 15
F V MMF 8 15 16 F MMF 2 16 15 F HCQ, MMF 0 17 15 F 120 HCQ 6 18 16
F 3 HCQ, MMF 4 19 15 F R 10 HCQ, MMF 4 20 17 F A, R 10 MMF 10 21 14
F 10 MMF 5 22 17 M R HCQ 10 23 14 F HCQ 4 24 10 F R 5 HCQ 6 25 17 F
C HCQ 6 26 11 F 10 HCQ, MMF 0 27 13 F R HCQ, MMF 6 28 16 F HCQ 2 29
12 F 10 HCQ, MMF 8 30 17 F R 10 HCQ, MPA 10 31 15 F A 10 HCQ, MMF 4
32 16 M 10 HCQ, MMF 4 33 15 M R 10 HCQ, MMF, MTX 8 34 17 F R 10 HCQ
8 35 16 F 10 HCQ, MMF 4 36 16 F R 10 HCQ, MMF, MTX 8 37 17 F 2 HCQ,
MMF 2 38 16 F 5 MMF 4 Abbreviations: F: female, M: male, A:
articular, C: cutaneous, D: digestive, H: haematologic, M:
mycardic, N: neurologic, PP: pleuro-pericardic, R: renal, V:
vascular, APS: anti-phopholipid syndrome, HCQ: hydroxychloroquin,
AZA: azathioprin, CYC: cyclophosphamide, MMF: mycophenolate
mofetil, MPA: mycophenolic acid, MTX: methotrexate, RTX:
rituximab.
Skin and Kidney biopsies (class IV lupus nephritis) were randomly
selected in regard to patient characteristics from the adult SLE
population. Control skin samples were obtained from patients
undergoing plastic surgery. Control kidney samples were obtained
from cancer patients who underwent nephrectomy.
Phenotyping of Blood Immune Cells by Flow Cytometry--
For the analysis of OX40L expression, whole blood samples were
stained with anti-CD14-PC5, CD16-FITC, CD11c-APC, HLA-DR-PC7, and
OX40L-PE mAbs, and red blood cells were lysed with Versalyse
(Beckman Coulter). For the analysis of blood Tfh cells, whole blood
samples were stained with anti-CXCR5-AF488, CCR6-PE, CXCR3-PC5,
CCR4-PC7, CD3-AF700, CD8-APCH7, CD4-Pacific Blue (all from Becton
Dickinson), CD45RA-ECD (Beckman Coulter), ICOS-APC (Biolegend) and
CD45-Pacific Orange (Invitrogen). Data were collected using a BD
LSR II instrument (BD Biosciences) and analyzed with Flowjo
software (Tree Star Inc.).
Skin and Kidney Biopsy Analysis--
OX40L and CD11c expression in skin and kidney biopsies from SLE
patients and subjects without autoimmune diseases was analyzed
using immunofluorescence microscopy. Briefly, 5-.mu.m-thick
sections of formalin-fixed, paraffin-embedded tissues from skin and
kidney were deparaffinized and subjected to a heat-induced epitope
retrieval step. Slides were rinsed in cool running water and washed
in Tris-buffered saline, pH 7.4, before overnight incubation with
primary anti-CD11c (Novocsatra, clone 5D11), and anti-OX40L
(R&D Systems, clone 159403) or isotype-matched control
antibody. Slides were then washed three times and incubated with
appropriate secondary antibodies: AF488-conjugated anti-mouse or
rat or AF568-conjugated anti-mouse (Invitrogen). Immunofluorescence
image was analyzed on an Olympus BX51/52 system microscope coupled
to a Cytovision System (Applied Imaging).
Phenotypic Analysis of Tonsil Samples--
Tonsil samples were obtained from healthy subjects undergoing
tonsillectomies, and single cells were collected by mechanical
disruption. For surface staining, cells were incubated with
fluorochrome conjugated antibodies CD11c-PC7 (B-ly6), HLA-DR-PerCP
(L243), CD19-FITC (HIB19), CD19-APC (HIB19) and 41BBL-PE (C65-485)
from BD Biosciences, OX40L-PE (ANC10G1) from Ancell Corporation,
CD14-APC-AF750 (TuK4), CD16-APC (3G8) and CD3-PB (UCHT1) from
Invitrogen, ICOSL-FITC (MIH12) from Miltenyi, GITRL-PE (109101)
from R&D in the presence of LIVE/DEAD FIXABLE Aqua (Invitrogen)
for 15 minutes, followed by analysis on the BD LSRII. Data were
further analysed using FlowJo software (Tree Star Inc.).
Culture of Th Cells--
Naive (CD45RA.sup.+CCR7.sup.+) and memory (CD45RA.sup.-) Th cells
were sorted by flow cytometry as described before (Schmitt et al.,
2009). Th cells were stimulated overnight with CD3/CD28 Dynabeads
(Invitrogen) in RPMI complete medium supplemented with 10% FCS.
Cells were then transferred to flat-bottomed 96 well plates coated
with CD3 mAb (5 .mu.g/ml, OKT3) supplemented with soluble CD28 mAb
(1 .mu.g/ml. CD28.2), in the presence or absence of recombinant
IL-12 (100 pg/ml), and/or soluble OX40L (100 ng/ml (R&D
systems). In some experiments, sorted naive Th cells were cultured
with allogeneic monocytes (CD14.sup.+ cells) isolated by sort from
active SLE patient PBMCs (T: monocyte ratio=1:1). T cells were
harvested day 4 (for CD3/CD28 stimulated T cells) or at day 7 (for
monocyte-T co-culture) for phenotyping with anti-CXCR5-AF647,
anti-CD40L APC-eFluor 780 and anti-ICOS biotin/Streptavidin-PerCP;
and for co-culture with B cells. For the assessment of IL-21
expression (with anti-IL21-PE), cultured cells were re-stimulated
with 25 ng/ml PMA, 1 .mu.g/ml ionomycin for 6 hours in the presence
of Brefeldine (eBioscience) and monensin for the last 4 hours.
Co-Culture of Th and B Cells--
Activated Th cells were co-cultured with autologous naive or memory
B cells (5.times.10.sup.3 T cells for 40.times.10.sup.3 memory B
cells per well) in 96-well round-bottom plates in Yssel medium/10%
FBS in the presence of endotoxin-reduced SEB (0.25 ng/ml; Toxin
technology, Inc.). IgG produced in the cultures were analyzed by
ELISA at day 14.
Culture of Monocytes--
CD14.sup.+ monocytes were purified from blood samples from healthy
donors by negative selection (Schmitt et al., 2009) and then
exposed to SLE serum (10%) or control serum for 3 days in a 6
well-plate. The phenotype was analyzed by FACS with anti-CD14-PC5,
anti-HLA-DR-PC7, and anti-OX40L-PE. TLR3 (poly-IC, 10 .mu.g/ml),
TLR7 (R837, 5 .mu.g/ml), TLR9 (ODN2216, 10 .mu.g/ml) agonists were
purchased from InvivoGen. The TLR7 inhibitor IRS-661 (1
.mu.M)(Barrat et al., 2005) was incubated for 10 min with the
monocytes before with the addition of SLE serum or anti-RNP IgG (50
.mu.g/ml).
Anti-RNP Purification--
Anti-RNP titer levels were measured using commercially available
enzyme-linked immunosorbant assay (ELISA) kits (Corgenix). Samples
were compared with a positive control provided by the manufacturer.
Positive anti-RNP samples had an activity greater than 22 U/ml. IgG
were purified from anti-RNP.sup.+ SLE patients' serum samples using
HiTrap Protein G HP column (GE Healthcare). Purified IgG were
desalted and then quantified.
Nanostring--
Th cells cultured with IL-12 and/or sOX40L for 48 h were lysed in
RLT buffer. Total RNA was purified using RNeasy Micro Kit (Qiagen).
The NanoString reactions were done according to manufacturer's
instructions. The data were normalized to housekeeping genes
included in the codeset.
Statistical Analysis--
The normality of the variable distribution was assessed and as the
normality of the distribution was rejected, analyses were performed
by the non-parametric paired Wilcoxon test or unpaired Mann-Whitney
U tests as appropriate. When necessary, comparisons were analyzed
with Kruskall Wallis test followed by Dunn post hoc test. To
compare more than three parameters, one-way ANOVA with multiple
comparison tests was used. Correlation between variables was
determined by using the Spearman test. All statistical analyses
were performed using StatView 5.0 software (SAS Institute).
Results
OX40L is Abundantly Expressed in Inflamed Tonsils--
Applicants previously demonstrated that dermal CD14+ DCs
preferentially induce the generation of Tfh-like cells in vitro
(Klechevsky et al., 2008). Skin DC subsets including dermal CD14+
DCs migrate into the draining lymph nodes (Segura et al., 2012),
and interact with T cells at the T cell zone. CD206+ DCs in the
lymph nodes, a proposed counterpart of migrating dermal CD14+ DCs,
promote naive Th cells to produce CXCL13 (Segura et al., 2012), the
chemokine abundantly expressed by Tfh cells (Bentebibel et al.,
2011; Kim et al., 2004). These observations suggest the involvement
of dermal CD14+ DCs in the generation of Tfh cells and antibody
responses in draining lymph nodes of skin. However, the phenotype
of APCs associated with Tfh responses in inflammatory lymphoid
organs such as tonsils has not been determined.
Previous studies in mouse models demonstrated the importance of
ICOS ligand (ICOSL) expressed by DCs for the differentiation of Tfh
cells (Choi et al., 2011). Applicants analyzed whether myeloid APCs
(CD11c+HLA-DR+) express ICOSL in pediatric tonsils, which are
enriched with mature Tfh cells along with GCs (Bentebibel et al.,
2011). Applicants found that myeloid APCs in inflamed tonsils
barely expressed ICOSL (FIG. 9A, 9B). Instead myeloid APCs, in
particular CD14+ cells, expressed the co-stimulatory molecule OX40L
(9.3.+-.7.1% of CD11c+HLA-DR+ cells, Mean.+-.s.d., n=9. FIG. 9A).
Other TNF ligand family molecules such as GITRL and 4-1BBL were
undetectable or expressed only at low levels (FIG. 9 A, B).
However, OX40L expression by myeloid APCs was nearly absent in
spleen (0.3.+-.0.5% of CD11c+HLA-DR+ cells, Mean.+-.s.d., n=4),
where Tfh and GC responses are much less evident than in pediatric
tonsils (Bentebibel et al., 2013). Thus, OX40L+ myeloid APCs are
not present in all the secondary lymphoid organs, and appear to be
restricted to those with inflammation.
To determine their localization, tonsil tissues were stained with
anti-OX40L and anti-CD11c and analyzed by immunofluorescence
microscopy. Applicants found that OX40L was abundantly expressed in
tonsils, in particular subepithelial area, T cell zones, and mantle
zones; but less in GCs (FIG. 9C). OX40L.sup.+ CD11c.sup.+ myeloid
APCs were mainly found in the T cell zone (FIG. 9C). It is notable
that OX40L was also expressed by CD11c.sup.- cells. This is
consistent with the fact that OX40L can be expressed by a broad
range of immune cells including B cells, vascular endothelial
cells, mast cells, activated NK cells, and activated Th cells
(Croft, 2010). These observation suggests that inflammatory
environment in tonsils induces upregulation of OX40L expression on
different types of cells by unknown mechanisms.
Myeloid APCs from Active Adult and Pediatric SLE Patients Express
OX40L--
Given prominent expression of OX40L in inflamed tonsils, Applicants
wondered whether OX40L was also expressed in inflammatory tissues
from SLE patients. Applicants found that OX40L was abundantly
expressed by cells including CD11c.sup.+ myeloid APCs in
inflammatory kidney tissues from active adult SLE patients with
nephritis, but absent in tissues from subjects without autoimmune
diseases (FIG. 10A). OX40L.sup.+ myeloid APCs were also found in
skin biopsy samples from SLE patients, but not from controls (FIG.
10A). Similar to tonsils, OX40L.sup.+ CD11c.sup.- cells were also
present in both tissues from SLE patients.
Applicants next analyzed whether peripheral myeloid APCs in
patients with SLE also express OX40L. OX40L expression was
significantly increased on the surface of blood myeloid APCs from
adult and pediatric patients with active SLE compared to healthy
subjects, inactive SLE patients, and other autoimmune disease
patients (FIG. 12B, and FIG. 15). Similar to tonsillar myeloid APCs
(FIG. 9A, 9B), the expression of ICOSL, GITRL, or 4-1BBL on blood
myeloid APCs was not observed (FIG. 16). The percentage of
OX40L.sup.+ cells in blood was significantly higher in active
patients (assessed by the SLE Disease Activity Index (SLEDAI)) than
in inactive patients, both in adult and pediatric SLE (FIG. 10C).
Furthermore, the frequency of OX40L.sup.+ cells within myeloid APCs
correlated with disease activity as assessed by the SLEDAI in both
adult and pediatric SLE (FIG. 10D). OX40L was mainly expressed by
CD14.sup.+CD16.sup.+ and CD14.sup.+CD16.sup.- monocytes (FIG. 10E,
and FIG. 17). In a longitudinal follow-up of 11 flaring and
previously untreated adult SLE patients, the percentage of
OX40L.sup.+ myeloid APC substantially decreased after treatment
along with the decrease in disease activity (FIG. 18, P<0.01,
n=1).
Taken together, these results show that OX40L is expressed on blood
and tissue-infiltrating myeloid APCs in active SLE patients.
OX40 Signals Promote the Expression of Tfh Genes in Naive and
Memory T Cells--
The presence of OX40L.sup.+ myeloid APCs in blood and inflamed
tissues suggests that OX40L expression is globally increased on
myeloid APCs in active SLE patients. In particular, inflamed
tissues in SLE patients appear to create an OX40L-rich environment
where Th cells receive OX40 signals together with T cell receptor
signals (FIG. 10A). While providing signals important for T cell
proliferation and survival, OX40 signals also regulate Th
differentiation in collaboration with other factors derived from
APCs, microenvironment, and Th cells themselves (Croft, 2010).
Applicants hypothesized that OX40 signals might display an
intrinsic property to promote the differentiation of human Th cells
towards the Tfh lineage. To address this hypothesis, applicants
applied an APC-free system to avoid the contribution of factors
from APCs and microenvironment, and cultured naive and memory Th
cells with anti-CD3 and anti-CD28 in the presence of agonistic
soluble OX40L (sOX40L). To minimize the influence of T
cell-intrinsic factors, applicants analyzed the gene expression
profiles at 48 h of culture by NanoString. For the assessment of
the impact of OX40 signals on gene expression patterns, the
transcript abundance in Th cells stimulated in the presence of
sOX40L was normalized to the values in Th cells stimulated in the
absence of sOX40L. Applicants found that OX40 signaling upregulated
multiple Tfh genes, including CXCR5, BCL6, IL21, CXCL13, and PDCD1
(encoding PD-1) in both naive and memory Th cells (FIG. 11A).
Furthermore, OX40L stimulation downregulated the expression of
PRDM1 (encoding Blimp-1), the transcription repressor that inhibits
Tfh generation (Crotty, 2011).
Previously applicants and others show that IL-12 induces activated
human naive Th cells to express multiple Tfh molecules including
IL-21, ICOS, CXCR5, and Bcl-6 at higher levels than other cytokines
(Ma et al., 2009; Schmitt et al., 2013; Schmitt et al., 2009).
Subjects deficient of IL-12 receptor .beta.1 (IL-12R .beta.1) chain
display reduced Tfh and GC responses (in particular children),
providing in vivo evidence that signals via IL-12 receptor is
essential for the generation of Tfh cell differentiation in humans
(Schmitt et al., 2013). Thus, applicants compared the expression of
Tfh genes between OX40- and IL-12-stimulated Th cells.
Surprisingly, OX40 signals induced naive Th cells to express Tfh
genes at equivalent levels with IL-12 signals (FIG. 19).
Furthermore, overall expression patterns of Tfh genes were largely
similar between OX40- and IL-12-stimulated naive Th cells (FIG.
11B, left). While mouse studies suggest the positive role of
IFN-.gamma. for the generation of Tfh cells (Lee et al., 2012), the
upregulation of Tfh molecules in these cells was not due to
IFN-.gamma. secreted in the cultures, as IFN-.gamma.-stimulated
naive Th cells did not show the similar gene patterns (FIG. 19B,
left). The combination of the two signals further increased the
expression of IL21, but not other Tfh molecules.
In contrast to the observation with naive Th cells, OX40 signals
were more potent than IL-12 signals at inducing memory Th cells to
modulate the expression of global Tfh genes (FIG. 11B, right) and
at promoting the upregulation of Tfh genes (BCL6, CXCR5, IL-21,
CXCL13, and PDCD1), and the downregulation of PRDM1 gene (FIG.
11C). It was notable that OX40 signals differentially modulated the
expression of MAF and BATF, genes associated with Tfh development
and functions (Crotty, 2011), between naive and memory Th cells.
OX40 signals induced upregulation of the two genes in naive Th
cells, but downregulation in memory Th cells (FIG. 11B).
Nonetheless, IL-12 signals cooperated with OX40 signals to increase
the expression of IL21 by memory Th cells (FIG. 11C).
OX40 Signals Promote the Generation of Functional Helpers--
To analyze the expression of Tfh molecules at protein levels, naive
and memory Th cells were activated by anti-CD3 and anti-CD28 in the
presence or absence of sOX40L for three days, and the phenotype was
analyzed by flow cytometry. Consistent with transcriptional data
(FIG. 11A, 11B), OX40 signals promoted both naive and memory Th
cells to express Tfh molecules including CXCR5, CD40L, and IL-21,
and increased the generation of CXCR5.sup.+ cells co-expressing
IL-21, CD40L and ICOS (FIGS. 12A and 12B; FIG. 20). Of note, in
addition to IL-21, OX40 signals induced the expression of IL-2 and
TNF-.alpha., but not IFN-.gamma. or IL-4 (FIG. 21). Furthermore,
OX40L stimulation induced naive Th cells to downregulate the
expression of CCR7 on CXCR5.sup.+ cells (FIG. 20), and increased
the generation of CXCR5+CCR7.sup.- cells, a chemokine receptor
expression profile required for homing to B cell follicles (Haynes
et al., 2007). Strikingly, OX40 signals induced memory Th cells to
express Tfh molecules including CXCR5, CD40L, and IL-21 more
efficiently than IL-12 signals (FIG. 12B). Applicants noticed that
OX40 signals decreased the expression of ICOS on memory Th cells
compared to the control culture (FIG. 20), which was consistent
with the transcriptional data (FIG. 11B). However, ICOS expression
levels remained high, and more than 80% of CXCR5.sup.+ cells
stimulated with OX40 signals expressed ICOS.
Applicants wondered whether OX40 signals are sufficient to induce
Th cells to become functional helpers. To this end, stimulated Th
cells were co-cultured with autologous B cells and the produced IgG
were measured at day 14. OX40 signals were sufficient to induce
both naive and memory Th cells to become B cell helpers (FIG. 12C).
Notably, OX40 signals were more efficient than IL-12 signals to
induce memory Th cells to become helpers (FIG. 12C). These results
show that OX40L stimulation promotes naive and memory Th cells to
differentiate into Tfh-like cells.
Collectively, these results show that OX40 signals display an
intrinsic property to induce human naive and memory Th cells to
express multiple Tfh molecules and to become functional B cell
helpers.
Myeloid APCs from SLE Patients Promote the Generation of
IL-21.sup.+ Th Cells in an OX40L Dependent Manner--
These data suggest that OX40 signals contribute to the generation
of aberrant Tfh response in SLE. Thus, applicants examined whether
blood OX40L.sup.+ myeloid APCs from active SLE patients induce Th
cells to express Tfh molecules. Applicants used total CD14.sup.+
monocytes for the experiments as isolation of OX40L.sup.+ monocytes
was not feasible. CD14.sup.+ monocytes were isolated from active
and inactive adult SLE patients and cultured with allogeneic naive
Th cells. Applicants found that CD14.sup.+ monocytes from active
patients induced naive Th cells to become CXCR5.sup.+IL-21.sup.+ T
cells more efficiently than those from inactive SLE patients (FIG.
13A). The generation of CXCR5.sup.+IL-21.sup.+ cells was largely
dependent on OX40L, because blocking the OX40-OX40L interactions
with a OX40L-neutralizing mAb strongly inhibited it (FIGS. 13B and
13C). Furthermore, the number of the generated
CXCR5.sup.+IL-21.sup.+ cells positively correlated with the
frequency of OX40L.sup.+ monocytes in the cultures (FIG. 13D).
Previous studies showed that active SLE patients display an
increased frequency of blood Tfh cells with active phenotype
(ICOS.sup.+CXCR5.sup.+) (He et al., 2013; Simpson et al., 2010).
Applicants were able to confirm this observation in the cohort
(FIG. 13E). Applicants further found that the frequency of ICOS+
cells within blood Tfh cells positively correlated with the
frequency of OX40L+ cells within blood myeloid APCs (FIG. 13F). The
frequency of OX40L+ APCs also positively correlated with the
frequency of blood Tfh cells (CXCR5+ in total Th cells) (FIG. 22),
but showed no correlation with the frequency of blood CXCR5- Th1
(CXCR3+CCR6-), Th2 (CXCR3-CCR6-) Th17 (CXCR3-CCR6+) cells (Morita
et al., 2011) (FIG. 23). Collectively, these results suggest that
OX40L- expressing myeloid APCs from SLE patients promote the
development and/or the activation of Tfh cells.
RNP/anti-RNP ICs promote OX40L expression through TLR7 activation.
Applicants wondered which mechanism is involved in OX40L expression
by myeloid APCs in active SLE patients. Applicants previously
demonstrated that SLE sera induce monocytes to acquire the
properties of DCs (Blanco et al., 2001). Therefore, applicants
hypothesized that SLE sera might contain components that induce
OX40L expression by myeloid APCs. Accordingly, applicants found
that SLE sera, but not control sera, induced OX40L expression on
normal monocytes at variable levels (FIG. 14A). Applicants
suspected the involvement of immune complexes (ICs) containing self
nucleic acid, because activation of APCs through endosomal nucleic
acid sensors play a key role in SLE pathogenesis (Barrat and
Coffman, 2008). Indeed, stimulation with agonist of TLR7, but not
TLR9 and 3, induced normal monocytes to express OX40L (FIG. 14B).
To test whether TLR7 was directly implicated in OX40L upregulation
by SLE sera, applicants exposed normal monocytes to SLE sera in the
presence of a specific TLR7 inhibitor IRS-661 (Barrat et al., 2005)
or RNAse. Both TLR7 inhibitor and RNAse significantly reduced the
ability of SLE sera to induce OX40L expression (FIG. 14C; FIG. 24),
suggesting the major role by ICs containing RNA. In agreement with
this hypothesis, applicants observed that the presence of
anti-ribonucleoprotein (RNP), but not anti-DNA, antibodies in SLE
sera was associated with the increased ability to promote OX40L
expression on normal monocytes (FIG. 14D).
To validate whether RNP/anti-RNP ICs were directly involved in
OX40L expression, monocytes were cultured with anti-RNP negative
SLE sera, and purified IgG containing RNP/anti-RNP ICs were spiked
into the cultures. Applicants found that the supplementation with
RNP/anti-RNP ICs rendered anti-RNP negative SLE sera able to
promote OX40L expression (FIG. 14E). This effect was dependent on
TLR7, as addition of TLR7-specific inhibitor abrogated the
upregulation of OX40L (FIG. 14E).
These data show that RNP/anti-RNP ICs promote OX40L expression
through TLR7 activation in myeloid APCs in active SLE.
Autoreactive antibody production is a hallmark of a variety of
autoimmune diseases including SLE. This study provides evidence
that OX40L expressed by myeloid APCs contributes to lupus
pathogenesis by promoting the generation of Tfh cells.
The expression of OX40L by myeloid APCs was increased in blood as
well as in inflammatory tissues in active SLE patients. OX40L.sup.+
myeloid APCs in blood of active SLE patients were largely confined
to CD14.sup.+CD16.sup.- and CD14.sup.+CD16.sup.+ monocyte
populations. OX40L.sup.+ myeloid APCs in pediatric tonsils were
also largely limited to the CD14.sup.+ population. Increased OX40L
expression on blood monocyte populations was also reported in
patients with sepsis (Karulf et al., 2010) and patients with
chronic hepatitis C (Zhang et al., 2013). Interestingly, both
disease conditions are known to be often associated with hyper
gammaglobulinemia. These observations suggest that monocytes and
macrophages upregulate OX40L in inflammatory environment, and
contribute to antibody responses.
The pathogenic roles of ICs containing self nucleic acid are well
established in SLE. The ICs activate plasmacytoid DCs via TLR9 and
TLR7, and induce them to produce large amounts of type I
interferons (Lovgren et al., 2006). Type I IFN stimulation induces
neutrophils to upregulate TLR7, and renders them able to respond to
RNP/anti-RNP ICs. Then neutrophils produce DNA-containing
components that activate pDCs (Garcia-Romo et al., 2011; Lande et
al., 2011). RNP/anti-RNP ICs also target the CD16.sup.+CD14.sup.dim
monocyte population, and induce these cells to produce cytokines
that damage the endothelium, including TNF-.alpha., IL-1, and CCL3
(Cros et al., 2010). While these mechanisms activate the innate
immune system and cause inflammation, this study shows that
RNP/anti-RNP ICs also activate the adaptive immune system. The
inventors found that RNP/anti-RNP ICs contribute to OX40L
expression by monocytes/macrophages via TLR7. Tfh responses
increased by the RNP/anti-RNP IC-OX40L axis further accelerate the
generation of autoantibodies including those against self nucleic
acid. Therefore, the RNP/anti-RNP IC-OX40L axis appear to provide
an amplification loop of the generation of autoantibodies in
SLE.
The inventors showed that OX40 signals together with TCR and CD28
signals promote naive and memory Th cells to express multiple Tfh
molecules, including CXCR5, IL-21, and Bcl-6, while suppressing the
expression of Blimp-1. The inventors also found that OX40 signals
and IL-12 signals were almost equivalently efficient at inducing
human naive Th cells to express Tfh molecules. Furthermore, the two
signals cooperate in the upregulation of IL-21 expression by Th
cells. Remarkably, OX40 signals were more potent than IL-12 signals
to induce memory Th cells to express Tfh molecules, and were
sufficient to render them to become efficient B cell helpers. These
results show that OX40 signals display intrinsic property to drive
Th differentiation towards the Tfh lineage. It is presumable that
interactions with OX40L-expressing APCs in inflammatory tissues in
SLE render memory T cells to differentiate into Tfh cells and
thereby potentially perpetuate the B cell autoimmune response in
situ. Nonetheless, reduced Tfh and GC responses in subjects
deficient of ICOS, CD40L (Bossaller et al., 2006), IL-12R.beta.1
chain (Schmitt et al., 2013), and STAT3 (Ma et al., 2012) suggest
that OX40 signals by themselves are not sufficient to compensate
the lack of these signals. Furthermore, subjects deficient of OX40
show intact antibody responses in vivo despite less blood memory B
cells (Byun et al., 2013), indicating that OX40 signals can be
dispensable for generation of antibody responses. Therefore, the
inventors surmise that excessive OX40 signals cause aberrant Tfh
response and autoimmunity in humans. The positive correlation
between the frequency of ICOS.sup.+ blood Tfh cells and the
frequency of OX40L.sup.+ myeloid APCs in active SLE patients
supports this hypothesis.
Early mouse studies showed that OX40L stimulation promotes mouse
naive Th cells to express CXCR5 (Flynn et al., 1998), and their
migration into B cell follicles (Brocker et al., 1999; Fillatreau
and Gray, 2003). Furthermore, an OX40L-transgenic mice model (T
cell-specific overexpression) showed development an autoimmune-like
disease characterized by interstitial pneumonia, colitis, and high
levels of anti-nuclear antibodies (Murata et al., 2002). Recent
studies show that the mutation of Roquin gene in sanroque mice
causes upregulation of OX40 on Th cells, suggesting the positive
role of OX40 signals for the generation of Tfh cells (Pratama et
al., 2013; Vogel et al., 2013). On the other hand, at least two
studies concluded that the absence of OX40 signals did not affect
CXCR5 expression by Th cells, Tfh differentiation, GC development,
or antibody generation (Akiba et al., 2005; Kopf et al., 1999).
Furthermore, in vivo treatment with agonistic OX40 mAb inhibited
Tfh cell generation in mice in an acute viral infection model
(Boettler et al., 2013) and in a listeria infection model (Marriott
et al., 2014. Boettler et al. showed that agonistic anti-OX40 mAb
induced enhanced the expression of Blimp-1 by specific Th cells
while suppressing the expression of Bcl-6 in vivo (Boettler et al.,
2013), contrary to the inventors' observations with human Th cells
in vitro. Given that OX40 signals regulate Th differentiation in
collaboration with other factors derived from APCs,
microenvironment, and Th cells themselves (Croft, 2010), it is
possible that OX40 signals promote or suppress Tfh cell
differentiation according to the microenvironment where Th cells
interact with APCs. Another possibility is that OX40 signals
differentially induce Tfh molecules between human and mouse Th
cells.
The inventors' conclusion is also supported by the findings in GWAS
in autoimmune diseases. TNFSF4 (encoding OX40L) polymorphism has
been found to confer susceptibility to SLE (Cunninghame Graham et
al., 2008; Delgado-Vega et al., 2009) and other autoimmune
diseases, such as Sjogren syndrome, and rheumatoid arthritis (Kim
et al., 2014; Nordmark et al., 2011). The TNFSF4 risk allele is
also associated with renal disorder in Caucasian SLE patients
(Sanchez et al., 2011). Furthermore, copy number variations and/or
polymorphism at the TLR7 locus has been shown to associate with SLE
susceptibility (Shen et al., 2010). This study provides a rationale
that therapeutic modalities targeting the RNP-containing
IC-OX40L-OX40 axis and TLR7 could impact the development of
autoantibodies and therefore be beneficial for human SLE.
Example 3: In Vivo Efficacy of Anti-OX40L in the Suppression of
GVHD
Allograft survival with no adverse effects is an ultimate goal in
transplantation. Over the past several decades, a large array of
immunosuppressive agents have been developed and used for patients
who underwent transplantation surgery. However, such
immunosuppressive drugs do not guarantee the prevention of
alloreaction over time in patients who receive organs, tissues, and
hematopoietic stem cell (HPSC) transplantation. As a consequence,
patients succumb to graft-versus-host disease (GVHD) as well as
serious side effects from life-long immunosuppression.
Therefore, the development of novel therapeutic strategies that
prevent GVHD without diminishing host immunity to microbial
pathogens is of particular importance. Anti-OX40L antibodies did
not interfere with human chimerism.
Anti-OX40L antibodies did not interfere with human chimerism (FIG.
26A-B). It was then tested whether anti-OX40L antibody (clone 19A3,
IgG2b) treatment could result in the suppression of GVHD in animal
model. In xenogeneic GVHD mouse models, human T cells activated
with human DCs are the major causes of diseases. Fifteen NOG mice
were .gamma.-irradiated one day before human PBMC (10 million
cells, intravenously) transplantation. Animals were divided into
three groups (five-six mice/group) and then treated with 200 .mu.g
of qan isotype control, 2 .mu.g anti-OX40L (clone 19A3), or 200
.mu.g anti-OX40L (clone 19A3) three times per week. FIG. 25 shows
that all animals treated with 200 .mu.g anti-OX40L were still alive
at day 112 whereas all the animals treated with the isotype control
had died (or had to be sacrificed due to adverse health reasons) by
day 42. Two-thirds of the animals treated with 2 .mu.g anti-OX40L
had died by day 112, and one-third was still alive. These data
indicate that anti-OX40L can suppress GVHD in vivo.
All of the methods disclosed and claimed herein can be made and
executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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SEQUENCE LISTINGS
1
4211420DNAMus musculus 1atggaatgga gctggatctt tctcttcctc ctgtcaggaa
ctgcaggtgt acactctgag 60gtccagcttc agcagtctgg gcctgagctg gggcagcctg
gggcttcagt gaagatatcc 120tgcaaggctt ctggttactc attcactggt
tacagcatgc actgggtgaa gcagagccat 180aggaaaagcc ctgagtggat
tggaaaaatt gatccttaca atggtgtgac tacctataat 240cagaggttca
cgggcaaggc cacattgact gtcgacacat cttccagcac agcctacatg
300catctcaaca gcctgacatc tgaggactct gcaatctttt actgtgcgag
agaggggttt 360gcttattggg gccaagggac tctggtctct gtctctgaag
ccaaaacaac acccccatca 420gtctatccac tggcccctgg gtgtggagat
acaactggtt cctccgtgac tctgggatgc 480ctggtcaagg gctacttccc
tgagtcagtg actgtgactt ggaactctgg atccctgtcc 540agcagtgtgc
acacmttccc agctctcctg cagtctggac tctacactat gagcagctca
600gtgactgtcc cctccagcac ctggccaagt cagaccgtca cctgcagcgt
tgctcaccca 660gccagcagca ccacggtgga caaaaaactt gagcccagcg
ggcccatttc aacaatcaac 720ccctgtcctc catgcaagga gtgtcacaaa
tgcccagctc ctaacctcga gggtggacca 780tccgtcttca tcttccctcc
aaatatcaag gatgtactca tgatctccct gacacccaag 840gtcacgtgtg
tggtggtgga tgtgagcgag gatgacccag acgtccagat cagctggttt
900gtgaacaacg tggaagtaca cacagctcag acacaaaccc atagagagga
ttacaacagt 960actatccggg tggtcagcac cctccccatc cagcaccagg
actggatgag tggcaaggag 1020ttcaaatgca aggtcaacaa caaagacctc
ccatcaccca tcgagagaac catctcaaaa 1080attaaagggc tagtcagagc
tccacaagta tacatcttgc cgccaccagc agagcagttg 1140tccaggaaag
atgtcagtct cacttgcctg gtcgtgggct tcaaccctgg agacatcagt
1200gtggagtgga ccagcaatgg gcatacagag gagaactaca aggacaccgc
accagtcctg 1260gactctgacg gttcttactt catatatagc aagctcaata
tgaaaacaag caagtgggag 1320aaaacagatt ccttctcatg caacgtgaga
cacgagggtc tgaaaaatta ctacctgaag 1380aagaccatct cccggtctcc
gggtaaagct agctgaaaaa 14202471PRTMus musculus 2Met Glu Trp Ser Trp
Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Gly Gln 20 25 30 Pro
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40
45 Thr Gly Tyr Ser Met His Trp Val Lys Gln Ser His Arg Lys Ser Pro
50 55 60 Glu Trp Ile Gly Lys Ile Asp Pro Tyr Asn Gly Val Thr Thr
Tyr Asn 65 70 75 80 Gln Arg Phe Thr Gly Lys Ala Thr Leu Thr Val Asp
Thr Ser Ser Ser 85 90 95 Thr Ala Tyr Met His Leu Asn Ser Leu Thr
Ser Glu Asp Ser Ala Ile 100 105 110 Phe Tyr Cys Ala Arg Glu Gly Phe
Ala Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Ser Val Ser Glu Ala
Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 130 135 140 Ala Pro Gly Cys
Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys 145 150 155 160 Leu
Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser 165 170
175 Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser
180 185 190 Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser
Thr Trp 195 200 205 Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro
Ala Ser Ser Thr 210 215 220 Thr Val Asp Lys Lys Leu Glu Pro Ser Gly
Pro Ile Ser Thr Ile Asn 225 230 235 240 Pro Cys Pro Pro Cys Lys Glu
Cys His Lys Cys Pro Ala Pro Asn Leu 245 250 255 Glu Gly Gly Pro Ser
Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val 260 265 270 Leu Met Ile
Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val 275 280 285 Ser
Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 290 295
300 Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser
305 310 315 320 Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln
Asp Trp Met 325 330 335 Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
Lys Asp Leu Pro Ser 340 345 350 Pro Ile Glu Arg Thr Ile Ser Lys Ile
Lys Gly Leu Val Arg Ala Pro 355 360 365 Gln Val Tyr Ile Leu Pro Pro
Pro Ala Glu Gln Leu Ser Arg Lys Asp 370 375 380 Val Ser Leu Thr Cys
Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser 385 390 395 400 Val Glu
Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr 405 410 415
Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu 420
425 430 Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys
Asn 435 440 445 Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys
Thr Ile Ser 450 455 460 Arg Ser Pro Gly Lys Ala Ser 465 470
319PRTMus musculus 3Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser
Gly Thr Ala Gly 1 5 10 15 Val His Ser 4116PRTMus musculus 4Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Gly Gln Pro Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20
25 30 Ser Met His Trp Val Lys Gln Ser His Arg Lys Ser Pro Glu Trp
Ile 35 40 45 Gly Lys Ile Asp Pro Tyr Asn Gly Val Thr Thr Tyr Asn
Gln Arg Phe 50 55 60 Thr Gly Lys Ala Thr Leu Thr Val Asp Thr Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu
Asp Ser Ala Ile Phe Tyr Cys 85 90 95 Ala Arg Glu Gly Phe Ala Tyr
Trp Gly Gln Gly Thr Leu Val Ser Val 100 105 110 Ser Glu Ala Lys 115
510PRTMus musculus 5Gly Tyr Ser Phe Thr Gly Tyr Ser Met His 1 5 10
617PRTMus musculus 6Lys Ile Asp Pro Tyr Asn Gly Val Thr Thr Tyr Asn
Gln Arg Phe Thr 1 5 10 15 Gly 75PRTMus musculus 7Glu Gly Phe Ala
Tyr 1 5 8712DNAMus musculus 8atgcattttc aagtgcagat tttcagcttc
ctgctaatca gtgcctcagt cataatgtcc 60agaggacaaa ttgttctcac ccagtctcca
gcaatcatgt ctgcatctcc aggggagaag 120gtcaccataa cctgcagtgc
ctcctcaagt gtccgttata ttcactggtt ccagcagaag 180ccaggcactt
ctcccaaact cttgatttat agcacatccg acctggcttc tggagtccct
240gctcgcttca gtggcggtgg atctgggacc tcttactctc tcacaatcag
ccgaatggag 300gctgaagatg ctgccactta ttactgccag caaaggactg
gttacccgct cacgttcggt 360gctgggacca agctggagct gaaacgggct
gatgctgcac caactgtatc catcttccca 420ccatccagtg agcagttaac
atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 480taccccaaag
acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc
540ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag
cagcaccctc 600acgttgacca aggacgagta tgaacgacat aacagctata
cctgtgaggc cactcacaag 660gcatcaactt cacccatcgt caagagcttc
aacaggaatg agtgttagaa aa 7129235PRTMus musculus 9Met His Phe Gln
Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile
Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser 35
40 45 Ser Ser Val Arg Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Thr
Ser 50 55 60 Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asp Leu Ala Ser
Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Met Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Arg 100 105 110 Thr Gly Tyr Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Arg Ala Asp Ala Ala
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 130 135 140 Gln Leu Thr
Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 145 150 155 160
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 165
170 175 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp
Ser 180 185 190 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp
Glu Tyr Glu 195 200 205 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His
Lys Ala Ser Thr Ser 210 215 220 Pro Ile Val Lys Ser Phe Asn Arg Asn
Glu Cys 225 230 235 1022PRTMus musculus 10Met His Phe Gln Val Gln
Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser
Arg Gly 20 11102PRTMus musculus 11Gln Ile Val Leu Thr Gln Ser Pro
Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr
Cys Ser Ala Ser Ser Ser Val Arg Tyr Ile 20 25 30 His Trp Phe Gln
Gln Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr 35 40 45 Ser Thr
Ser Asp Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Gly 50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65
70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Thr Gly Tyr Pro
Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys 100 1210PRTMus musculus
12Ser Ala Ser Ser Ser Val Arg Tyr Ile His 1 5 10 137PRTMus musculus
13Ser Thr Ser Asp Leu Ala Ser 1 5 149PRTMus musculus 14Gln Gln Arg
Thr Gly Tyr Pro Leu Thr 1 5 151404DNAArtificial SequenceSynthetic
Primer 15atgaagtgct cctgggtcat cttcttcctc atggccgtgg tgaccggagt
gaactctgag 60gtgcaactcc agcagtcagg agctgaaatc gtgaagccag gcgcaagtgt
gaagctgtcc 120tgcaccgctt ctgggttcaa catcaaggac acctacatgc
actgggtgaa gcagcggcca 180gagcaggggt tggagtggat tggcagaatt
gaccctagga acgacaacac caagtttgac 240cctaagtttc gcgggaaagc
aacactgact gccgatacat ccagcaatac tgcctacctg 300cagctgagca
gccttacatc cgaggatgcc gccgtctact actgtgtgcc cgtccccaca
360aggagctggt attttgatgt gtggggggcc ggcactagcg tcacagtctc
cagcgccaaa 420acaaagggcc catccgtctt ccccctggcg ccctgctcca
ggagcacctc cgagagcaca 480gccgccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc
660tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga
gtccaaatat 720ggtcccccat gcccaccctg cccagcacct gagttcgaag
ggggaccatc agtcttcctg 780ttccccccaa aacccaagga cactctcatg
atctcccgga cccctgaggt cacgtgcgtg 840gtggtggacg tgagccagga
agaccccgag gtccagttca actggtacgt ggatggcgtg 900gaggtgcata
atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg
960gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta
caagtgcaag 1020gtctccaaca aaggcctccc gtcctccatc gagaaaacca
tctccaaagc caaagggcag 1080ccccgagagc cacaggtgta caccctgccc
ccatcccagg aggagatgac caagaaccag 1140gtcagcctga cctgcctggt
caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
1260tccttcttcc tctacagcag gctaaccgtg gacaagagca ggtggcagga
ggggaatgtc 1320ttctcatgct ccgtgatgca tgaggctctg cacaaccact
acacacagaa gagcctctcc 1380ctgtctctgg gtaaagctag ctga
140416467PRTArtificial SequenceSynthetic Peptide 16Met Lys Cys Ser
Trp Val Ile Phe Phe Leu Met Ala Val Val Thr Gly 1 5 10 15 Val Asn
Ser Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Ile Val Lys 20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile 35
40 45 Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly
Leu 50 55 60 Glu Trp Ile Gly Arg Ile Asp Pro Arg Asn Asp Asn Thr
Lys Phe Asp 65 70 75 80 Pro Lys Phe Arg Gly Lys Ala Thr Leu Thr Ala
Asp Thr Ser Ser Asn 85 90 95 Thr Ala Tyr Leu Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ala Ala Val 100 105 110 Tyr Tyr Cys Val Pro Val Pro
Thr Arg Ser Trp Tyr Phe Asp Val Trp 115 120 125 Gly Ala Gly Thr Ser
Val Thr Val Ser Ser Ala Lys Thr Lys Gly Pro 130 135 140 Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165
170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr 225 230 235 240 Gly Pro Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 245 250 255 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 275 280 285
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290
295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys 340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 355 360 365 Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr 370 375 380 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410
415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
420 425 430 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 450 455 460 Lys Ala Ser 465 1719PRTArtificial
SequenceSynthetic Peptide 17Met Lys Cys Ser Trp Val Ile Phe Phe Leu
Met Ala Val Val Thr Gly 1 5 10 15 Val Asn Ser 18121PRTArtificial
SequenceSynthetic Peptide 18Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Ile Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Lys
Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp
Pro Arg Asn Asp Asn Thr Lys Phe Asp Pro Lys Phe 50 55 60 Arg Gly
Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ala Ala Val Tyr Tyr Cys 85
90 95 Val Pro Val Pro Thr Arg Ser Trp Tyr Phe Asp Val Trp Gly Ala
Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Ala Lys 115 120
1910PRTArtificial SequenceSynthetic Peptide 19Gly Phe Asn Ile Lys
Asp Thr Tyr Met His 1 5 10 2017PRTArtificial SequenceSynthetic
Peptide 20Arg Ile Asp Pro Arg Asn Asp Asn Thr Lys Phe Asp Pro Lys
Phe Arg 1 5 10 15 Gly 2110PRTArtificial SequenceSynthetic Peptide
21Val Pro Thr Arg Ser Trp Tyr Phe Asp Val 1 5 10 22717DNAArtificial
SequenceSynthetic Primer 22atggagaccc
attcccaagt gttcgtctac atgctgctct ggctctccgg agtcgaagga 60gacatcgtga
tgacccagtc tcacaagttc atgtccacca gcgtgggcga tagagtgtct
120attacctgca aggcctcaca ggacgtgggg aaatccgtcg tgtggtttca
gcagaagcct 180ggccagagtc caaagctttt gatctactgg gcaagcacca
ggcacacagg ggtgcccgat 240cggtttacag gcagcgggag cggcactgat
tttactctga caatttccaa cgtccagagc 300gaggacctgg ctaattattt
ctgtcagcag tacactagct acccctacat gtacacattc 360ggggggggca
caaagctcga gatcaaacga actgtggctg caccatctgt cttcatcttc
420ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct
gctgaataac 480ttctatccca gagaggccaa agtacagtgg aaggtggata
acgccctcca atcgggtaac 540tcccaggaga gtgtcacaga gcaggacagc
aaggacagca cctacagcct cagcagcacc 600ctgacgctga gcaaagcaga
ctacgagaaa cacaaagtct atgcctgcga agtcacccat 660cagggcctga
gctcgcccgt cacaaagagc ttcaacaggg gagagtgtgc tagctga
71723238PRTArtificial SequenceSynthetic Peptide 23Met Glu Thr His
Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser 1 5 10 15 Gly Val
Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser 20 25 30
Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 35
40 45 Val Gly Lys Ser Val Val Trp Phe Gln Gln Lys Pro Gly Gln Ser
Pro 50 55 60 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly
Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser 85 90 95 Asn Val Gln Ser Glu Asp Leu Ala Asn
Tyr Phe Cys Gln Gln Tyr Thr 100 105 110 Ser Tyr Pro Tyr Met Tyr Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160
Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165
170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys Ala Ser 225 230 235 2420PRTArtificial SequenceSynthetic
Peptide 24Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp
Leu Ser 1 5 10 15 Gly Val Glu Gly 20 25106PRTArtificial
SequenceSynthetic Peptide 25Gly Asp Ile Val Met Thr Gln Ser His Lys
Phe Met Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Ser Ile Thr Cys
Lys Ala Ser Gln Asp Val Gly Lys 20 25 30 Ser Val Val Trp Phe Gln
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu 35 40 45 Ile Tyr Trp Ala
Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln 65 70 75 80
Ser Glu Asp Leu Ala Asn Tyr Phe Cys Gln Gln Tyr Thr Ser Tyr Pro 85
90 95 Tyr Met Tyr Thr Phe Gly Gly Gly Thr Lys 100 105
2611PRTArtificial SequenceSynthetic Peptide 26Lys Ala Ser Gln Asp
Val Gly Lys Ser Val Val 1 5 10 277PRTArtificial SequenceSynthetic
Peptide 27Trp Ala Ser Thr Arg His Thr 1 5 2811PRTArtificial
SequenceSynthetic Peptide 28Gln Gln Tyr Thr Ser Tyr Pro Tyr Met Tyr
Thr 1 5 10 291416DNAArtificial SequenceSynthetic Primer
29atggagagac actggatctt gctcctgctg ttgtccgtga ccgctggagt ccatagccag
60gtccaactgc aacagtccgg agcagaactt gctaggcctg gagcaagcgt caaaatgtcc
120tgtaaggctt ccggatacac cctcgcaagc tacaccctgc actgggtgaa
gcagcgccct 180gggcaggggc ttgaatggat tggctatatt aatcccaaca
gtggctatac caactacatc 240cagaagttca aggacaaggc caccctcaca
gccgacaaga gctcatcaac tgcttacatg 300cagctgagtt ctctgacatc
tgaggacagt gccgtgtact actgcgctaa aggcggcggg 360gatcggtatt
gtacagattg cgccatggat tattggggcc agggcacatc tgtgactgtg
420tctcccgcca aaacaaaggg cccatccgtc ttccccctgg cgccctgctc
caggagcacc 480tccgagagca cagccgccct gggctgcctg gtcaaggact
acttccccga accggtgacg 540gtgtcgtgga actcaggcgc cctgaccagc
ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac tctactccct
cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 660aagacctaca
cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagagagtt
720gagtccaaat atggtccccc atgcccaccc tgcccagcac ctgagttcga
agggggacca 780tcagtcttcc tgttcccccc aaaacccaag gacactctca
tgatctcccg gacccctgag 840gtcacgtgcg tggtggtgga cgtgagccag
gaagaccccg aggtccagtt caactggtac 900gtggatggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gttcaacagc 960acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag
1020tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac
catctccaaa 1080gccaaagggc agccccgaga gccacaggtg tacaccctgc
ccccatccca ggaggagatg 1140accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctaccccag cgacatcgcc 1200gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260gactccgacg
gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag
1320gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacacag 1380aagagcctct ccctgtctct gggtaaagct agctga
141630471PRTArtificial SequenceSynthetic Peptide 30Met Glu Arg His
Trp Ile Leu Leu Leu Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His
Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg 20 25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu 35
40 45 Ala Ser Tyr Thr Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr
Asn Tyr Ile 65 70 75 80 Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala
Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Gly Gly
Gly Asp Arg Tyr Cys Thr Asp Cys Ala 115 120 125 Met Asp Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Pro Ala Lys 130 135 140 Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 145 150 155 160
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165
170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr 210 215 220 Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val 225 230 235 240 Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 245 250 255 Glu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 290
295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser 340 345 350 Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410
415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 450 455 460 Leu Ser Leu Gly Lys Ala Ser 465 470
3119PRTArtificial SequenceSynthetic Peptide 31Met Glu Arg His Trp
Ile Leu Leu Leu Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser
32125PRTArtificial SequenceSynthetic Peptide 32Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ala Ser Tyr 20 25 30 Thr
Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ile Gln Lys Phe
50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Gly Gly Asp Arg Tyr Cys Thr
Asp Cys Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Pro Ala Lys 115 120 125 3310PRTArtificial SequenceSynthetic
Peptide 33Gly Tyr Thr Leu Ala Ser Tyr Thr Leu His 1 5 10
3417PRTArtificial SequenceSynthetic Peptide 34Tyr Ile Asn Pro Asn
Ser Gly Tyr Thr Asn Tyr Ile Gln Lys Phe Lys 1 5 10 15 Asp
3514PRTArtificial SequenceSynthetic Peptide 35Gly Gly Gly Asp Arg
Tyr Cys Thr Asp Cys Ala Met Asp Tyr 1 5 10 36711DNAArtificial
SequenceSynthetic Primer 36atgcactccc ttgcacttct gttgagcctc
ttgctgctgt gcgtgagtga cagcagagct 60gagaccaccg tgacacagtc tcctgcctct
ctgtcaatga ccatcggaga aaaggtgacc 120atcaggtgca tgactagcat
cgacattgac gatgatatga actggtacca gcagaagcca 180ggggagcctc
caaagctgct gatttccgag ggaaagacac tccgccccgg ggtccccagt
240cggttttcca gctccgggta cggcactgac tttgtcttca ctattgagaa
catgctcagc 300gaggatgtgg ccgattacta ttgtctccaa agcgacaatc
tgcccttcac attcggctcc 360ggcacaaaac tcgagatcaa acgaactgtg
gctgcaccat ctgtcttcat cttcccgcca 420tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag
540gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag
caccctgacg 600ctgagcaaag cagactacga gaaacacaaa gtctatgcct
gcgaagtcac ccatcagggc 660ctgagctcgc ccgtcacaaa gagcttcaac
aggggagagt gtgctagctg a 71137236PRTArtificial SequenceSynthetic
Peptide 37Met His Ser Leu Ala Leu Leu Leu Ser Leu Leu Leu Leu Cys
Val Ser 1 5 10 15 Asp Ser Arg Ala Glu Thr Thr Val Thr Gln Ser Pro
Ala Ser Leu Ser 20 25 30 Met Thr Ile Gly Glu Lys Val Thr Ile Arg
Cys Met Thr Ser Ile Asp 35 40 45 Ile Asp Asp Asp Met Asn Trp Tyr
Gln Gln Lys Pro Gly Glu Pro Pro 50 55 60 Lys Leu Leu Ile Ser Glu
Gly Lys Thr Leu Arg Pro Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Ser
Ser Gly Tyr Gly Thr Asp Phe Val Phe Thr Ile Glu 85 90 95 Asn Met
Leu Ser Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser Asp 100 105 110
Asn Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 115
120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys Ala Ser 225 230 235
3820PRTArtificial SequenceSynthetic Peptide 38Met His Ser Leu Ala
Leu Leu Leu Ser Leu Leu Leu Leu Cys Val Ser 1 5 10 15 Asp Ser Arg
Ala 20 39103PRTArtificial SequenceSynthetic Peptide 39Glu Thr Thr
Val Thr Gln Ser Pro Ala Ser Leu Ser Met Thr Ile Gly 1 5 10 15 Glu
Lys Val Thr Ile Arg Cys Met Thr Ser Ile Asp Ile Asp Asp Asp 20 25
30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Pro Pro Lys Leu Leu Ile
35 40 45 Ser Glu Gly Lys Thr Leu Arg Pro Gly Val Pro Ser Arg Phe
Ser Ser 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Val Phe Thr Ile Glu
Asn Met Leu Ser 65 70 75 80 Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln
Ser Asp Asn Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys 100
4011PRTArtificial SequenceSynthetic Peptide 40Met Thr Ser Ile Asp
Ile Asp Asp Asp Met Asn 1 5 10 417PRTArtificial SequenceSynthetic
Peptide 41Glu Gly Lys Thr Leu Arg Pro 1 5 429PRTArtificial
SequenceSynthetic Peptide 42Leu Gln Ser Asp Asn Leu Pro Phe Thr 1
5
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